Neoantigen Identification for T-Cell Therapy

ABSTRACT

A method for identifying T-cells that are antigen-specific for at least one neoantigen that is likely to be presented on surfaces of tumor cells of a subject. Peptide sequences of tumor neoantigens are obtained by sequencing the tumor cells of the subject. The peptide sequences are input into a machine-learned presentation model to generate presentation likelihoods for the tumor neoantigens, each presentation likelihood representing the likelihood that a neoantigen is presented by an MHC allele on the surfaces of the tumor cells of the subject. A subset of the neoantigens is selected based on the presentation likelihoods. T-cells that are antigen-specific for at least one of the neoantigens in the subset are identified. These T-cells can be expanded for use in T-cell therapy. TCRs of these identified T-cells can also be sequenced and cloned into new T-cells for use in T-cell therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/US2018/049614, filed Sep. 5, 2018, which claims the benefit of U.S.Provisional Application No. 62/554,4286, filed Sep. 5, 2017, and claimsthe benefit of U.S. Provisional Application No. 62/579,734, filed Oct.31, 2017, and claims the benefit of U.S. Provisional Application No.62/644,191, filed Mar. 16, 2018, and claims the benefit of U.S.Provisional Application No. 62/703,197, filed Jul. 25, 2018, thecontents of which are hereby incorporated by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 2, 2018, isnamed GSO-037WOUS_CRF_sequencelisting.txt and is 71,751 bytes in size.

BACKGROUND

Therapeutic vaccines and T-cell therapy based on tumor-specificneoantigens hold great promise as a next-generation of personalizedcancer immunotherapy.¹⁻³ Cancers with a high mutational burden, such asnon-small cell lung cancer (NSCLC) and melanoma, are particularlyattractive targets of such therapy given the relatively greaterlikelihood of neoantigen generation.^(4,5) Early evidence shows thatneoantigen-based vaccination can elicit T-cell responses⁶ and thatneoantigen targeted T-cell therapy can cause tumor regression undercertain circumstances in selected patients.⁷ Both MHC class I and MHCclass II have an impact on T-cell responses⁷⁰⁻⁷¹.

However identification of neoantigens and neoantigen-recognizing T-cellshas become a central challenge in assessing tumor responses^(77,110),examining tumor evolution¹¹¹ and designing the next generation ofpersonalized therapies¹¹². Current neoantigen identification techniquesare either time-consuming and laborious^(84,96), or insufficientlyprecise^(87,91-93) Although it has recently been demonstrated thatneoantigen-recognizing T-cells are a major component ofTIL^(84,96,113,114) and circulate in the peripheral blood of cancerpatients¹⁰⁷, current methods for identifying neoantigen-reactive T-cellshave some combination of the following three limitations: (1) they relyon difficult-to-obtain clinical specimens such as TIL^(97,98) orleukaphereses¹⁰⁷ (2) they require screening impractically largelibraries of peptides⁹⁵ or (3) they rely on MHC multimers, which maypractically be available for only a small number of MHC alleles.

Furthermore, initial methods have been proposed incorporatingmutation-based analysis using next-generation sequencing, RNA geneexpression, and prediction of MHC binding affinity of candidateneoantigen peptides⁸. However, these proposed methods can fail to modelthe entirety of the epitope generation process, which contains manysteps (e.g., TAP transport, proteasomal cleavage, MHC binding, transportof the peptide-MHC complex to the cell surface, and/or TCR recognitionfor MHC-I; endocytosis or autophagy, cleavage via extracellular orlysosomal proteases (e.g., cathepsins), competition with the CLIPpeptide for HLA-DM-catalyzed HLA binding, transport of the peptide-MHCcomplex to the cell surface and/or TCR recognition for MHC-II) inaddition to gene expression and MHC binding⁹. Consequently, existingmethods are likely to suffer from reduced low positive predictive value(PPV). (FIG. 1A)

Indeed, analyses of peptides presented by tumor cells performed bymultiple groups have shown that <5% of peptides that are predicted to bepresented using gene expression and MHC binding affinity can be found onthe tumor surface MHC^(10,11) (FIG. 1B). This low correlation betweenbinding prediction and MHC presentation was further reinforced by recentobservations of the lack of predictive accuracy improvement ofbinding-restricted neoantigens for checkpoint inhibitor response overthe number of mutations alone.¹²

This low positive predictive value (PPV) of existing methods forpredicting presentation presents a problem for neoantigen-based vaccinedesign and for neoantigen-based T-cell therapy. If vaccines are designedusing predictions with a low PPV, most patients are unlikely to receivea therapeutic neoantigen and fewer still are likely to receive more thanone (even assuming all presented peptides are immunogenic). Similarly,if therapeutic T-cells are designed based on predictions with a low PPV,most patients are unlikely to receive T-cells that are reactive to tumorneoantigens and the time and physical resource cost of identifyingpredictive neoantigens using downstream laboratory techniquespost-prediction may be unduly high. Thus, neoantigen vaccination andT-cell therapy with current methods is unlikely to succeed in asubstantial number of subjects having tumors. (FIG. 1C)

Additionally, previous approaches generated candidate neoantigens usingonly cis-acting mutations, and largely neglected to consider additionalsources of neo-ORFs, including mutations in splicing factors, whichoccur in multiple tumor types and lead to aberrant splicing of manygenes¹³, and mutations that create or remove protease cleavage sites.

Finally, standard approaches to tumor genome and transcriptome analysiscan miss somatic mutations that give rise to candidate neoantigens dueto suboptimal conditions in library construction, exome andtranscriptome capture, sequencing, or data analysis. Likewise, standardtumor analysis approaches can inadvertently promote sequence artifactsor germline polymorphisms as neoantigens, leading to inefficient use ofvaccine capacity or auto-immunity risk, respectively.

SUMMARY

Disclosed herein is an optimized approach for identifying and selectingneoantigens for personalized cancer vaccines, for T-cell therapy, orboth. First, optimized tumor exome and transcriptome analysis approachesfor neoantigen candidate identification using next-generation sequencing(NGS) are addressed. These methods build on standard approaches for NGStumor analysis to ensure that the highest sensitivity and specificityneoantigen candidates are advanced, across all classes of genomicalteration. Second, novel approaches for high-PPV neoantigen selectionare presented to overcome the specificity problem and ensure thatneoantigens advanced for vaccine inclusion and/or as targets for T-celltherapy are more likely to elicit anti-tumor immunity. These approachesinclude, depending on the embodiment, trained statistical regression ornonlinear deep learning models that jointly model peptide-allelemappings as well as the per-allele motifs for peptide of multiplelengths, sharing statistical strength across peptides of differentlengths. The nonlinear deep learning models particularly can be designedand trained to treat different MHC alleles in the same cell asindependent, thereby addressing problems with linear models that wouldhave them interfere with each other. Finally, additional considerationsfor personalized vaccine design and manufacturing based on neoantigens,and for production of personalized neoantigen-specific T-cells forT-cell therapy, are addressed.

The model disclosed herein outperforms state-of-the-art predictorstrained on binding affinity and early predictors based on MS peptidedata by up to an order of magnitude. By more reliably predictingpresentation of peptides, the model enables more time- andcost-effective identification of neoantigen-specific or tumorantigen-specific T-cells for personlized therapy using a clinicallypractical process that uses limited volumes of patient peripheral blood,screens few peptides per patient, and does not necessarily rely on MHCmultimers. However, in another embodiment, the model disclosed hereincan be used to enable more time- and cost-effective identification oftumor antigen-specific T cells using MHC multimers, by decreasing thenumber of peptides bound to MHC multimers that need to be screened inorder to identify neoantigen- or tumor antigen-specific T cells

The predictive performance of the model disclosed herein on the TILneoepitope dataset and the prospective neoantigen-reactive T-cellidentification task demonstrate that it is now possible to obtaintherapeutically-useful neoepitope predictions by modeling HLA processingand presentation. In summary, this work offers practical in silicoantigen identification for antigen-targeted immunotherapy, therebyaccelerating progress towards cures for patients.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, and accompanying drawings, where:

FIG. 1A shows current clinical approaches to neoantigen identification.

FIG. 1B shows that <5% of predicted bound peptides are presented ontumor cells.

FIG. 1C shows the impact of the neoantigen prediction specificityproblem.

FIG. 1D shows that binding prediction is not sufficient for neoantigenidentification.

FIG. 1E shows probability of MHC-I presentation as a function of peptidelength.

FIG. 1F shows an example peptide spectrum generated from Promega'sdynamic range standard. FIG. 1F discloses SEQ ID NO: 1.

FIG. 1G shows how the addition of features increases the model positivepredictive value.

FIG. 2A is an overview of an environment for identifying likelihoods ofpeptide presentation in patients, in accordance with an embodiment.

FIGS. 2B and 2C illustrate a method of obtaining presentationinformation, in accordance with an embodiment. FIG. 2B discloses SEQ IDNO: 28. FIG. 2C discloses SEQ ID NOS 3-8, respectively, in order ofappearance.

FIG. 3 is a high-level block diagram illustrating the computer logiccomponents of the presentation identification system, according to oneembodiment.

FIG. 4 illustrates an example set of training data, according to oneembodiment. FIG. 4 discloses the “Peptide Sequences” as SEQ ID NOS 10-13and the “C-Flanking Sequences” as SEQ ID NOS 15, 29-30, and 30,respectively, in order of appearance.

FIG. 5 illustrates an example network model in association with an MHCallele.

FIG. 6A illustrates an example network model NN_(H)(⋅) shared by MHCalleles, according to one embodiment.

FIG. 6B illustrates an example network model NN_(H)(⋅) shared by MHCalleles, according to another embodiment.

FIG. 7 illustrates generating a presentation likelihood for a peptide inassociation with an MHC allele using an example network model.

FIG. 8 illustrates generating a presentation likelihood for a peptide inassociation with a MHC allele using example network models.

FIG. 9 illustrates generating a presentation likelihood for a peptide inassociation with MHC alleles using example network models.

FIG. 10 illustrates generating a presentation likelihood for a peptidein association with MHC alleles using example network models.

FIG. 11 illustrates generating a presentation likelihood for a peptidein association with MHC alleles using example network models.

FIG. 12 illustrates generating a presentation likelihood for a peptidein association with MHC alleles using example network models.

FIG. 13A illustrates a sample frequency distribution of mutation burdenin NSCLC patients.

FIG. 13B illustrates the number of presented neoantigens in simulatedvaccines for patients selected based on an inclusion criteria of whetherthe patients satisfy a minimum mutation burden, in accordance with anembodiment.

FIG. 13C compares the number of presented neoantigens in simulatedvaccines between selected patients associated with vaccines includingtreatment subsets identified based on presentation models and selectedpatients associated with vaccines including treatment subsets identifiedthrough current state-of-the-art models, in accordance with anembodiment.

FIG. 13D compares the number of presented neoantigens in simulatedvaccines between selected patients associated with vaccines includingtreatment subsets identified based on a single per-allele presentationmodel for HLA-A*02:01 and selected patients associated with vaccinesincluding treatment subsets identified based on both per-allelepresentation models for HLA-A*02:01 and HLA-B*07:02. The vaccinecapacity is set as t=20 epitopes, in accordance with an embodiment.

FIG. 13E compares the number of presented neoantigens in simulatedvaccines between patients selected based on mutation burden and patientsselected by expectation utility score, in accordance with an embodiment.

FIG. 14A compares the positive predictive values (PPV) at 40% recall ofthe “Full MS Model,” the “Peptide MS Model,” and the MHCFlurry 1.2.0binding affinity model with the three different gene expressionthresholds of TPM >0, 1, and 2 when each model is tested on a test setcomprising five different test samples, each test sample comprising aheld-out tumor sample with a 1:2500 ratio of presented to non-presentedpeptides.

FIG. 14B compares PPV at 40% recall of the “Full MS Model,” the “PeptideMS Model,” and the MHCFlurry 1.2.0 binding affnity model with the threedifferent gene expression thresholds of TPM >0, 1, and 2, when eachmodel is tested on a test set comprising 15 different test samples, eachtest sample comprising held-out peptides from a single-allele cell linetest dataset with a 1:10,000 ratio of presented to non-presentedpeptides.

FIG. 14C compares the proportion of somatic mutations recognized byT-cells (e.g., pre-existing T-cell responses) for the top 5, 10, and20-ranked somatic mutations identified by the “Full MS Model.” the“Peptide MS Model,” and the MHCFlurry 1.2.0 binding affinity model withthe three different gene expression thresholds of TPM >0, 1, and 2 for atest set comprising 12 different test samples, each test sample takenfrom a patient with at least one pre-existing T-cell response.

FIG. 14D compares the proportion of minimal neoepitopes recognized byT-cells (e.g., pre-existing T-cell responses) for the top 5, 10, and20-ranked minimal neoepitopes identified by the “Full MS Model,” the“Peptide MS Model,” and the MHCFlurry 1.2.0 binding affinity model withthe three different gene expression thresholds of TPM >0, 1, and 2 for atest set comprising 12 different test samples, each test sample takenfrom a patient with at least one pre-existing T-cell response.

FIG. 15A depicts detection of T-cell responses to patient-specificneoantigen peptide pools for nine patients.

FIG. 15B depicts detection of T-cell responses to individualpatient-specific neoantigen peptides for four patients.

FIG. 15C depicts example images of ELISpot wells for patient CU04.

FIG. 16 compares the positive predictive values (PPV) at 400% recall ofthe “Full MS Model” and an “Anchor Residue Only MS Model,” when eachmodel is tested on a test set comprising five different test samples,each test sample comprising a held-out tumor sample with a 1:2500 ratioof presented to non-presented peptides.

FIG. 17A depicts full precision-recall curves for the “Full MS Model,”the “Peptide MS Model,” and the MHCFlurry 1.2.0 binding affinity modelwith the three different gene expression thresholds of TPM >0, 1, and 2when each model is tested on test sample 0 from FIG. 14A.

FIG. 17B compares PPV at 40% recall of the “Full MS Model” the “PeptideMS Model,” and the MHCFlurry 1.2.0 binding affinity model with the threedifferent gene expression thresholds of TPM >0, 1, and 2, when eachmodel is tested on a test set comprising 15 different test samples, eachtest sample comprising held-out peptides from a single-allele cell linetest dataset with a 1:5,000 ratio of presented to non-presentedpeptides.

FIG. 17C depicts full precision-recall curves for the “Full MS Model,”the “Peptide MS Model,” and the MHCFlurry 1.2.0 binding affinity modelwith the three different gene expression thresholds of TPM >0, 1, and 2when each model is tested on test sample 0 from FIG. 14A.

FIG. 17D depicts full precision-recall curves for the “Full MS Model,”the “Peptide MS Model,” and the MHCFlurry 1.2.0 binding affinity modelwith the three different gene expression thresholds of TPM >0, 1, and 2when each model is tested on test sample 1 from FIG. 14A.

FIG. 17E depicts full precision-recall curves for the “Full MS Model,”the “Peptide MS Model,” and the MHCFlurry 1.2.0 binding affinity modelwith the three different gene expression thresholds of TPM >0, 1, and 2when each model is tested on test sample 2 from FIG. 14A.

FIG. 17F depicts full precision-recall curves for the “Full MS Model,”the “Peptide MS Model,” and the MHCFlurry 1.2.0 binding affinity modelwith the three different gene expression thresholds of TPM >0, 1, and 2when each model is tested on test sample 3 from FIG. 14A.

FIG. 17G depicts full precision-recall curves for the “Full MS Model,”the “Peptide MS Model,” and the MHCFlurry 1.2.0 binding affinity modelwith the three different gene expression thresholds of TPM >0, 1, and 2when each model is tested on test sample 4 from FIG. 14A.

FIG. 17H depicts full precision-recall curves for the “Full MS Model,”the “Peptide MS Model,” and the MHCFlurry 1.2.0 binding affinity modelwith the three different gene expression thresholds of TPM >0, 1, and 2when each model is tested on held-out peptides from the HLA-A*01:01 cellline test dataset from FIG. 14B with a 1:10,000 ratio of presented tonon-presented peptides.

FIG. 17I depicts full precision-recall curves for the “Full MS Model,”the “Peptide MS Model,” and the MHCFlurry 1.2.0 binding affinity modelwith the three different gene expression thresholds of TPM >0, 1, and 2when each model is tested on held-out peptides from the HLA-A*02:01 cellline test dataset from FIG. 14B with a 1:10,000 ratio of presented tonon-presented peptides.

FIG. 17J depicts full precision-recall curves for the “Full MS Model,”the “Peptide MS Model,” and the MHCFlurry 1.2.0 binding affinity modelwith the three different gene expression thresholds of TPM >0, 1, and 2when each model is tested on held-out peptides from the HLA-A*02:03 cellline test dataset from FIG. 14B with a 1:10,000 ratio of presented tonon-presented peptides.

FIG. 17K depicts full precision-recall curves for the “Full MS Model,”the “Peptide MS Model,” and the MHCFlurry 1.2.0 binding affinity modelwith the three different gene expression thresholds of TPM >0, 1, and 2when each model is tested on held-out peptides from the HLA-A*02:07 cellline test dataset from FIG. 14B with a 1:10,000 ratio of presented tonon-presented peptides.

FIG. 17L depicts full precision-recall curves for the “Full MS Model,”the “Peptide MS Model,” and the MHCFlurry 1.2.0 binding affinity modelwith the three different gene expression thresholds of TPM >0, 1, and 2when each model is tested on held-out peptides from the HLA-A*03:01 cellline test dataset from FIG. 14B with a 1:10,000 ratio of presented tonon-presented peptides.

FIG. 17M depicts full precision-recall curves for the “Full MS Model,”the “Peptide MS Model,” and the MHCFlurry 1.2.0 binding affinity modelwith the three different gene expression thresholds of TPM >0, 1, and 2when each model is tested on held-out peptides from the HLA-A*24:02 cellline test dataset from FIG. 14B with a 1:10,000 ratio of presented tonon-presented peptides.

FIG. 17N depicts full precision-recall curves for the “Full MS Model.”the “Peptide MS Model.” and the MHCFlurry 1.2.0 binding affinity modelwith the three different gene expression thresholds of TPM >0, 1, and 2when each model is tested on held-out peptides from the HLA-A*29:02 cellline test dataset from FIG. 14B with a 1:10,000 ratio of presented tonon-presented peptides.

FIG. 17O depicts full precision-recall curves for the “Full MS Model,”the “Peptide MS Model,” and the MHCFlurry 1.2.0 binding affinity modelwith the three different gene expression thresholds of TPM >0, 1, and 2when each model is tested on held-out peptides from the HLA-A*31:01 cellline test dataset from FIG. 14B with a 1:10,000 ratio of presented tonon-presented peptides.

FIG. 17P depicts full precision-recall curves for the “Full MS Model.”the “Peptide MS Model,” and the MHCFlurry 1.2.0 binding affinity modelwith the three different gene expression thresholds of TPM >0, 1, and 2when each model is tested on held-out peptides from the HLA-A*68:02 cellline test dataset from FIG. 14B with a 1:10,000 ratio of presented tonon-presented peptides.

FIG. 17Q depicts full precision-recall curves for the “Full MS Model,”the “Peptide MS Model,” and the MHCFlurry 1.2.0 binding affinity modelwith the three different gene expression thresholds of TPM >0, 1, and 2when each model is tested on held-out peptides from the HLA-A*35:01 cellline test dataset from FIG. 14B with a 1:10,000 ratio of presented tonon-presented peptides.

FIG. 17R depicts full precision-recall curves for the “Full MS Model,”the “Peptide MS Model,” and the MHCFlurry 1.2.0 binding affinity modelwith the three different gene expression thresholds of TPM >0, 1, and 2when each model is tested on held-out peptides from the HLA-A*44:02 cellline test dataset from FIG. 14B with a 1:10,000 ratio of presented tonon-presented peptides.

FIG. 17S depicts full precision-recall curves for the “Full MS Model,”the “Peptide MS Model,” and the MHCFlurry 1.2.0 binding affinity modelwith the three different gene expression thresholds of TPM >0, 1, and 2when each model is tested on held-out peptides from the HLA-A*44:03 cellline test dataset from FIG. 14B with a 1:10,000 ratio of presented tonon-presented peptides.

FIG. 17T depicts full precision-recall curves for the “Full MS Model,”the “Peptide MS Model,” and the MHCFlurry 1.2.0 binding affinity modelwith the three different gene expression thresholds of TPM >0, 1, and 2when each model is tested on held-out peptides from the HLA-A*51:01 cellline test dataset from FIG. 14B with a 1:10,000 ratio of presented tonon-presented peptides.

FIG. 17U depicts full precision-recall curves for the “Full MS Model,”the “Peptide MS Model,” and the MHCFlurry 1.2.0 binding affinity modelwith the three different gene expression thresholds of TPM >0, 1, and 2when each model is tested on held-out peptides from the HLA-A*54:01 cellline test dataset from FIG. 14B with a 1:10,000 ratio of presented tonon-presented peptides.

FIG. 17V depicts full precision-recall curves for the “Full MS Model,”the “Peptide MS Model,” and the MHCFlurry 1.2.0 binding affinity modelwith the three different gene expression thresholds of TPM >0, 1, and 2when each model is tested on held-out peptides from the HLA-A*57:01 cellline test dataset from FIG. 14B with a 1:10,000 ratio of presented tonon-presented peptides.

FIG. 18 compares the positive predictive values (PPV) at 40% recall ofdifferent versions of the MS Model and earlier approaches to modelingHLA presented peptides²⁹ in human tumors, when each model is tested onthe test set of FIG. 14A comprising five different test samples, eachtest sample comprising a held-out tumor sample with a 1:2500 ratio ofpresented to non-presented peptides.

FIG. 19A depicts results from control experiments with neoantigens inHLA-matched healthy donors.

FIG. 19B depicts results from control experiments with neoantigens inHLA-matched healthy donors. FIG. 19B discloses SEQ ID NOS 27, 24, 21-22,31-36, 21, 37-45, respectively, in order of appearance.

FIG. 20 depicts detection of T-cell responses to PHA positive controlfor each donor and each in vitro expansion depicted in FIG. 15A.

FIG. 21A depicts detection of T-cell responses to each individualpatient-specific neoantigen peptide in pool #2 for patient CU04.

FIG. 21B depicts detection of T-cell responses to individualpatient-specific neoantigen peptides for each of three visits of patientCU04 and for each of two visits of patient 1-024-002, each visitoccurring at a different time point.

FIG. 21C depicts detection of T-cell responses to individualpatient-specific neoantigen peptides and to patient-specific neoagntigenpeptide pools for each of two visits of patient CU04 and for each of twovisits of patient 1-024-002, each visit occurring at a different timepoint.

FIG. 22 depicts detection of T-cell responses to the twopatient-specific neoantigen peptide pools and to DMSO negative controlsfor the patients of FIG. 15A.

FIG. 23 compares the predictive performance of the “MS Model,”“NetMHCIIpan rank”: NetMHCIIpan 3.1⁷⁷, taking the lowest NetMHCIIpanpercentile rank across HLA-DRB1*15:01 and HLA-DRB5*01:01, and“NetMHCIIpan nM”: NetMHCIIpan 3.1, taking the strongest affinity in nMunits across HLA-DRB1*15:01 and HLA-DRB5*01:01, at ranking the peptidesin the HLA-DRB1*15:01/HLA-DRB5*01:01 test dataset.

FIG. 24 depicts a method for sequencing TCRs of neoantigen-specificmemory T-cells from the peripheral blood of a NSCLC patient. FIG. 24discloses SEQ ID NOS 46-48, respectively, in order of appearance.

FIG. 25 depicts exemplary embodiments of TCR constructs for introducinga TCR into recipient cells.

FIG. 26 depicts an exemplary P526 construct backbone nucleotide sequencefor cloning TCRs into expression systems for therapy development. FIG.26 discloses SEQ ID NO: 49.

FIG. 27 depicts an exemplary construct sequence for cloning patientneoantigen-specific TCR, clonotype 1 TCR into expression systems fortherapy development. FIG. 27 discloses SEQ ID NO: 50.

FIG. 28 depicts an exemplary construct sequence for cloning patientneoantigen-specific TCR, clonotype 3 into expression systems for therapydevelopment. FIG. 28 discloses SEQ ID NO: 51.

FIG. 29 is a flow chart of a method for providing a customized,neoantigen-specific treatment to a patient, in accordance with anembodiment.

FIG. 30 illustrates an example computer for implementing the entitiesshown in FIGS. 1 and 3.

DETAILED DESCRIPTION I. Definitions

In general, terms used in the claims and the specification are intendedto be construed as having the plain meaning understood by a person ofordinary skill in the art. Certain terms are defined below to provideadditional clarity. In case of conflict between the plain meaning andthe provided definitions, the provided definitions are to be used.

As used herein the term “antigen” is a substance that induces an immuneresponse.

As used herein the term “neoantigen” is an antigen that has at least onealteration that makes it distinct from the corresponding wild-type,parental antigen, e.g., via mutation in a tumor cell orpost-translational modification specific to a tumor cell. A neoantigencan include a polypeptide sequence or a nucleotide sequence. A mutationcan include a frameshift or nonframeshift indel, missense or nonsensesubstitution, splice site alteration, genomic rearrangement or genefusion, or any genomic or expression alteration giving rise to a neoORF.A mutations can also include a splice variant. Post-translationalmodifications specific to a tumor cell can include aberrantphosphorylation. Post-translational modifications specific to a tumorcell can also include a proteasome-generated spliced antigen. See Liepeet al., A large fraction of HLA class I ligands are proteasome-generatedspliced peptides; Science. 2016 Oct. 21; 354(6310):354-358.

As used herein the term “tumor neoantigen” is a neoantigen present in asubject's tumor cell or tissue but not in the subject's correspondingnormal cell or tissue.

As used herein the term “neoantigen-based vaccine” is a vaccineconstruct based on one or more neoantigens, e.g., a plurality ofneoantigens.

As used herein the term “candidate neoantigen” is a mutation or otheraberration giving rise to a new sequence that may represent aneoantigen.

As used herein the term “coding region” is the portion(s) of a gene thatencode protein.

As used herein the term “coding mutation” is a mutation occurring in acoding region.

As used herein the term “ORF” means open reading frame.

As used herein the term “NEO-ORF” is a tumor-specific ORF arising from amutation or other aberration such as splicing.

As used herein the term “missense mutation” is a mutation causing asubstitution from one amino acid to another.

As used herein the term “nonsense mutation” is a mutation causing asubstitution from an amino acid to a stop codon.

As used herein the term “frameshift mutation” is a mutation causing achange in the frame of the protein.

As used herein the term “indel” is an insertion or deletion of one ormore nucleic acids.

As used herein, the term percent “identity,” in the context of two ormore nucleic acid or polypeptide sequences, refer to two or moresequences or subsequences that have a specified percentage ofnucleotides or amino acid residues that are the same, when compared andaligned for maximum correspondence, as measured using one of thesequence comparison algorithms described below (e.g., BLASTP and BLASTNor other algorithms available to persons of skill) or by visualinspection. Depending on the application, the percent “identity” canexist over a region of the sequence being compared. e.g., over afunctional domain, or, alternatively, exist over the full length of thetwo sequences to be compared.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters. Alternatively, sequence similarity ordissimilarity can be established by the combined presence or absence ofparticular nucleotides, or, for translated sequences, amino acids atselected sequence positions (e.g., sequence motifs).

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information.

As used herein the term “non-stop or read-through” is a mutation causingthe removal of the natural stop codon.

As used herein the term “epitope” is the specific portion of an antigentypically bound by an antibody or T-cell receptor.

As used herein the term “immunogenic” is the ability to elicit an immuneresponse, e.g., via T-cells, B cells, or both.

As used herein the term “HLA binding affinity” “MHC binding affinity”means affinity of binding between a specific antigen and a specific MHCallele.

As used herein the term “bait” is a nucleic acid probe used to enrich aspecific sequence of DNA or RNA from a sample.

As used herein the term “variant” is a difference between a subject'snucleic acids and the reference human genome used as a control.

As used herein the term “variant call” is an algorithmic determinationof the presence of a variant, typically from sequencing.

As used herein the term “polymorphism” is a germline variant. i.e., avariant found in all DNA-bearing cells of an individual.

As used herein the term “somatic variant” is a variant arising innon-germline cells of an individual.

As used herein the term “allele” is a version of a gene or a version ofa genetic sequence or a version of a protein.

As used herein the term “HLA type” is the complement of HLA genealleles.

As used herein the term “nonsense-mediated decay” or “NMD” is adegradation of an mRNA by a cell due to a premature stop codon.

As used herein the term “truncal mutation” is a mutation originatingearly in the development of a tumor and present in a substantial portionof the tumor's cells.

As used herein the term “subclonal mutation” is a mutation originatinglater in the development of a tumor and present in only a subset of thetumor's cells.

As used herein the term “exome” is a subset of the genome that codes forproteins. An exome can be the collective exons of a genome.

As used herein the term “logistic regression” is a regression model forbinary data from statistics where the logit of the probability that thedependent variable is equal to one is modeled as a linear function ofthe dependent variables.

As used herein the term “neural network” is a machine learning model forclassification or regression consisting of multiple layers of lineartransformations followed by element-wise nonlinearities typicallytrained via stochastic gradient descent and back-propagation.

As used herein the term “proteome” is the set of all proteins expressedand/or translated by a cell, group of cells, or individual.

As used herein the term “peptidome” is the set of all peptides presentedby MHC-I or MHC-II on the cell surface. The peptidome may refer to aproperty of a cell or a collection of cells (e.g., the tumor peptidome,meaning the union of the peptidomes of all cells that comprise thetumor).

As used herein the term “ELISPOT” means Enzyme-linked immunosorbent spotassay—which is a common method for monitoring immune responses in humansand animals.

As used herein the term “dextramers” is a dextran-based peptide-MHCmultimers used for antigen-specific T-cell staining in flow cytometry.

As used herein the term “MHC multimers” is a peptide-MHC complexcomprising multiple peptide-MHC monomer units.

As used herein the term “MHC tetramers” is a peptide-MHC complexcomprising four peptide-MHC monomer units.

As used herein the term “tolerance or immune tolerance” is a state ofimmune non-responsiveness to one or more antigens, e.g. self-antigens.

As used herein the term “central tolerance” is a tolerance affected inthe thymus, either by deleting self-reactive T-cell clones or bypromoting self-reactive T-cell clones to differentiate intoimmunosuppressive regulatory T-cells (Tregs).

As used herein the term “peripheral tolerance” is a tolerance affectedin the periphery by downregulating or anergizing self-reactive T-cellsthat survive central tolerance or promoting these T-cells todifferentiate into Tregs.

The term “sample” can include a single cell or multiple cells orfragments of cells or an aliquot of body fluid, taken from a subject, bymeans including venipuncture, excretion, ejaculation, massage, biopsy,needle aspirate, lavage sample, scraping, surgical incision, orintervention or other means known in the art.

The term “subject” encompasses a cell, tissue, or organism, human ornon-human, whether in vivo, ex vivo, or in vitro, male or female. Theterm subject is inclusive of mammals including humans.

The term “mammal” encompasses both humans and non-humans and includesbut is not limited to humans, non-human primates, canines, felines,murines, bovines, equines, and porcines.

The term “clinical factor” refers to a measure of a condition of asubject, e.g., disease activity or severity. “Clinical factor”encompasses all markers of a subject's health status, includingnon-sample markers, and/or other characteristics of a subject, such as,without limitation, age and gender. A clinical factor can be a score, avalue, or a set of values that can be obtained from evaluation of asample (or population of samples) from a subject or a subject under adetermined condition. A clinical factor can also be predicted by markersand/or other parameters such as gene expression surrogates. Clinicalfactors can include tumor type, tumor sub-type, and smoking history.

Abbreviations: MHC: major histocompatibility complex; HLA: humanleukocyte antigen, or the human MHC gene locus; NGS: next-generationsequencing; PPV: positive predictive value; TSNA: tumor-specificneoantigen: FFPE: formalin-fixed, paraffin-embedded; NMD:nonsense-mediated decay; NSCLC: non-small-cell lung cancer: DC:dendritic cell.

It should be noted that, as used in the specification and the appendedclaims, the singular forms “a.” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

Any terms not directly defined herein shall be understood to have themeanings commonly associated with them as understood within the art ofthe invention. Certain terms are discussed herein to provide additionalguidance to the practitioner in describing the compositions, devices,methods and the like of aspects of the invention, and how to make or usethem. It will be appreciated that the same thing may be said in morethan one way. Consequently, alternative language and synonyms may beused for any one or more of the terms discussed herein. No significanceis to be placed upon whether or not a term is elaborated or discussedherein. Some synonyms or substitutable methods, materials and the likeare provided. Recital of one or a few synonyms or equivalents does notexclude use of other synonyms or equivalents, unless it is explicitlystated. Use of examples, including examples of terms, is forillustrative purposes only and does not limit the scope and meaning ofthe aspects of the invention herein.

All references, issued patents and patent applications cited within thebody of the specification are hereby incorporated by reference in theirentirety, for all purposes.

II. Methods of Identifying Neoantigens

Disclosed herein are methods for identifying T-cells that areantigen-specific for neoantigens from tumor cells of a subject that arelikely to be presented on a surface of the tumor cells. The methodincludes obtaining exome, transcriptome, and/or whole genome nucleotidesequencing data from the tumor cells as well as normal cells of thesubject. This nucleotide sequencing data is used to obtain a peptidesequence of each neoantigen in a set of neoantigens. The set ofneoantigens is identified by comparing the nucleotide sequencing datafrom the tumor cells and the nucleotide sequencing data from the normalcells. Specifically, the peptide sequence of each neoantigen in the setof neoantigens comprises at least one alteration that makes it distinctfrom the corresponding wild-type peptide sequence identified from thenormal cells of the subject. The method further includes encoding thepeptide sequence of each neoantigen in the set of neoantigens into acorresponding numerical vector. Each numerical vector includesinformation describing the amino acids that make up the peptide sequenceand the positions of the amino acids in the peptide sequence. The methodfurther comprises inputting the numerical vectors into a machine-learnedpresentation model to generate a presentation likelihood for eachneoantigen in the set of neoantigens. Each presentation likelihoodrepresents the likelihood that the corresponding neoantigen is presentedby MHC alleles on the surface of the tumor cells of the subject. Themachine-learned presentation model comprises a plurality of parametersand a function. The plurality of parameters are identified based on atraining data set. The training data set comprises, for each sample in aplurality of samples, a label obtained by mass spectrometry measuringpresence of peptides bound to at least one MHC allele in a set of MHCalleles identified as present in the sample, and training peptidesequences encoded as numerical vectors that include informationdescribing the amino acids that make up the peptides and the positionsof the amino acids in the peptides. The function represents a relationbetween the numerical vector received as input by the machine-learnedpresentation model and the presentation likelihood generated as outputby the machine-learned presentation model based on the numerical vectorand the plurality of parameters. The method further includes selecting asubset of the set of neoantigens, based on the presentation likelihoods,to generate a set of selected neoantigens. The method further comprisesidentifying T-cells that are antigen-specific for at least one of theneoantigens in the subset, and returning these identified T-cells.

In some embodiments, inputting the numerical vector into themachine-learned presentation model comprises applying themachine-learned presentation model to the peptide sequence of theneoantigen to generate a dependency score for each of the MHC alleles.The dependency score for an MHC allele indicates whether the MHC allelewill present the neoantigen, based on the particular amino acids at theparticular positions of the peptide sequence. In further embodiments,inputting the numerical vector into the machine-learned presentationmodel further comprises transforming the dependency scores to generate acorresponding per-allele likelihood for each MHC allele indicating alikelihood that the corresponding MHC allele will present thecorresponding neoantigen, and combining the per-allele likelihoods togenerate the presentation likelihood of the neoantigen. In someembodiments, transforming the dependency scores models the presentationof the neoantigen as mutually exclusive across the MHC alleles. Inalternative embodiments, inputting the numerical vector into themachine-learned presentation model further comprises transforming acombination of the dependency scores to generate the presentationlikelihood. In such embodiments, transforming the combination of thedependency scores models the presentation of the neoantigen asinterfering between the MHC alleles.

In some embodiments, the set of presentation likelihoods are furtheridentified by one or more allele noninteracting features. In suchembodiments, the method further comprises applying the machine-learnedpresentation model to the allele noninteracting features to generate adependency score for the allele noninteracting features. The dependencyscore indicates whether the peptide sequence of the correspondingneoantigen will be presented based on the allele noninteractingfeatures. In some embodiments, the method further comprises combiningthe dependency score for each MHC allele with the dependency score forthe allele noninteracting features, transforming the combined dependencyscore for each MHC allele to generate a per-allele likelihood for eachMHC allele, and combining the per-allele likelihoods to generate thepresentation likelihood. The per-allele likelihood for a MHC alleleindicates a likelihood that the MHC allele will present thecorresponding neoantigen. In alternative embodiments, the method furthercomprises combining the dependency scores for the MHC alleles and thedependency score for the allele noninteracting features, andtransforming the combined dependency scores to generate the presentationlikelihood.

In some embodiments, the MHC alleles include two or more different MHCalleles.

In some embodiments, the peptide sequences comprise peptide sequenceshaving lengths other than 9 amino acids.

In some embodiments, encoding the peptide sequence comprises encodingthe peptide sequence using a one-hot encoding scheme.

In some embodiments, the plurality of samples comprise at least one ofcell lines engineered to express a single MHC allele, cell linesengineered to express a plurality of MHC alleles, human cell linesobtained or derived from a plurality of patients, fresh or frozen tumorsamples obtained from a plurality of patients, and fresh or frozentissue samples obtained from a plurality of patients.

In some embodiments, the training data set further comprises at leastone of data associated with peptide-MHC binding affinity measurementsfor at least one of the peptides, and data associated with peptide-MHCbinding stability measurements for at least one of the peptides.

In some embodiments, the set of presentation likelihoods are furtheridentified by expression levels of the MHC alleles in the subject, asmeasured by RNA-seq or mass spectrometry.

In some embodiments, the set of presentation likelihoods are furtheridentified by features comprising at least one of predicted affinitybetween a neoantigen in the set of neoantigens and the MHC alleles, andpredicted stability of the neoantigen encoded peptide-MHC complex.

In some embodiments, the set of numerical likelihoods are furtheridentified by features comprising at least one of the C-terminalsequences flanking the neoantigen encoded peptide sequence within itssource protein sequence, and the N-terminal sequences flanking theneoantigen encoded peptide sequence within its source protein sequence.

In some embodiments, selecting the set of selected neoantigens comprisesselecting neoantigens that have an increased likelihood of beingpresented on the tumor cell surface relative to unselected neoantigens,based on the machine-learned presentation model.

In some embodiments, selecting the set of selected neoantigens comprisesselecting neoantigens that have an increased likelihood of being capableof inducing a tumor-specific immune response in the subject relative tounselected neoantigens, based on the machine-learned presentation model.

In some embodiments, selecting the set of selected neoantigens comprisesselecting neoantigens that have an increased likelihood of being capableof being presented to nave T-cells by professional antigen presentingcells (APCs) relative to unselected neoantigens, based on thepresentation model. In such embodiments, the APC is optionally adendritic cell (DC).

In some embodiments, selecting the set of selected neoantigens comprisesselecting neoantigens that have a decreased likelihood of being subjectto inhibition via central or peripheral tolerance relative to unselectedneoantigens, based on the machine-learned presentation model.

In some embodiments, selecting the set of selected neoantigens comprisesselecting neoantigens that have a decreased likelihood of being capableof inducing an autoimmune response to normal tissue in the subjectrelative to unselected neoantigens, based on the machine-learnedpresentation model.

In some embodiments, the one or more tumor cells are selected from thegroup consisting of: lung cancer, melanoma, breast cancer, ovariancancer, prostate cancer, kidney cancer, gastric cancer, colon cancer,testicular cancer, head and neck cancer, pancreatic cancer, braincancer. B-cell lymphoma, acute myelogenous leukemia, chronic myelogenousleukemia, chronic lymphocytic leukemia, and T-cell lymphocytic leukemia,non-small cell lung cancer, and small cell lung cancer.

In some embodiments, the method further comprises generating an outputfor constructing a personalized cancer vaccine from the set of selectedneoantigens. In such embodiments, the output for the personalized cancervaccine may comprise at least one peptide sequence or at least onenucleotide sequence encoding the set of selected neoantigens.

In some embodiments, the machine-learned presentation model is a neuralnetwork model. In such embodiments, the neural network model may includea plurality of network models for the MHC alleles, each network modelassigned to a corresponding MHC allele of the MHC alleles and includinga series of nodes arranged in one or more layers. In such embodiments,the neural network model may be trained by updating the parameters ofthe neural network model, the parameters of at least two network modelsbeing jointly updated for at least one training iteration. In someembodiments, the machine-learned presentation model may be a deeplearning model that includes one or more layers of nodes.

In some embodiments, identifying the T-cells comprises co-culturing theT-cells with one or more of the neoantigens in the subset underconditions that expand the T-cells.

In some embodiments, identifying the T-cells comprises contacting theT-cells with an MHC multimer comprising one or more of the neoantigensin the subset under conditions that allow binding between the T-cellsand the MHC multimer.

In some embodiments, the method further comprises identifying T-cellreceptors (TCR) of the identified T-cells. In such embodiments,identifying the T-cell receptors may comprise sequencing the T-cellreceptor sequences of the identified T-cells. In such embodiments, themethod may further comprise genetically engineering T-cells to expressat least one of the one or more identified T-cell receptors, culturingthe T-cells under conditions that expand the T-cells, and infusing theexpanded T-cells into the subject. In such embodiments, geneticallyengineering the T-cells to express at least one of the identified T-cellreceptors may comprise cloning the T-cell receptor sequences of theidentified T-cells into an expression vector, and transfecting each ofthe T-cells with the expression vector.

In some embodiments, the method further comprises culturing theidentified T-cells under conditions that expand the identified T-cells,and infusing the expanded T-cells into the subject.

In some embodiments, the T-cells that are antigen-specific for at leastone of the neoantigens in the subset are identified using between 5-30mL of whole blood from the subject.

In some embodiments, the subset of neoantigens comprises at most 20neoantigens and the identified T-cells recognize at least 2 neoantigensin the subset of neoantigens.

In some embodiments, the MHC alleles are class I MHC alleles.

Disclosed herein is also an isolated T-cell that is antigen-specific forat least one selected neoantigen in the subset of neoantigens describedabove.

III. Identification of Tumor Specific Mutations in Neoantigens

Also disclosed herein are methods for the identification of certainmutations (e.g., the variants or alleles that are present in cancercells). In particular, these mutations can be present in the genome,transcriptome, proteome, or exome of cancer cells of a subject havingcancer but not in normal tissue from the subject.

Genetic mutations in tumors can be considered useful for theimmunological targeting of tumors if they lead to changes in the aminoacid sequence of a protein exclusively in the tumor. Useful mutationsinclude: (1) non-synonymous mutations leading to different amino acidsin the protein; (2) read-through mutations in which a stop codon ismodified or deleted, leading to translation of a longer protein with anovel tumor-specific sequence at the C-terminus; (3) splice sitemutations that lead to the inclusion of an intron in the mature mRNA andthus a unique tumor-specific protein sequence; (4) chromosomalrearrangements that give rise to a chimeric protein with tumor-specificsequences at the junction of 2 proteins (i.e., gene fusion); (5)frameshift mutations or deletions that lead to a new open reading framewith a novel tumor-specific protein sequence. Mutations can also includeone or more of nonframeshift indel, missense or nonsense substitution,splice site alteration, genomic rearrangement or gene fusion, or anygenomic or expression alteration giving rise to a neoORF.

Peptides with mutations or mutated polypeptides arising from forexample, splice-site, frameshift, readthrough, or gene fusion mutationsin tumor cells can be identified by sequencing DNA, RNA or protein intumor versus normal cells.

Also mutations can include previously identified tumor specificmutations. Known tumor mutations can be found at the Catalogue ofSomatic Mutations in Cancer (COSMIC) database.

A variety of methods are available for detecting the presence of aparticular mutation or allele in an individual's DNA or RNA.Advancements in this field have provided accurate, easy, and inexpensivelarge-scale SNP genotyping. For example, several techniques have beendescribed including dynamic allele-specific hybridization (DASH),microplate array diagonal gel electrophoresis (MADGE), pyrosequencing,oligonucleotide-specific ligation, the TaqMan system as well as variousDNA “chip” technologies such as the Affymetrix SNP chips. These methodsutilize amplification of a target genetic region, typically by PCR.Still other methods, based on the generation of small signal moleculesby invasive cleavage followed by mass spectrometry or immobilizedpadlock probes and rolling-circle amplification. Several of the methodsknown in the art for detecting specific mutations are summarized below.

PCR based detection means can include multiplex amplification of aplurality of markers simultaneously. For example, it is well known inthe art to select PCR primers to generate PCR products that do notoverlap in size and can be analyzed simultaneously. Alternatively, it ispossible to amplify different markers with primers that aredifferentially labeled and thus can each be differentially detected. Ofcourse, hybridization based detection means allow the differentialdetection of multiple PCR products in a sample. Other techniques areknown in the art to allow multiplex analyses of a plurality of markers.

Several methods have been developed to facilitate analysis of singlenucleotide polymorphisms in genomic DNA or cellular RNA. For example, asingle base polymorphism can be detected by using a specializedexonuclease-resistant nucleotide, as disclosed, e.g., in Mundy, C. R.(U.S. Pat. No. 4,656,127). According to the method, a primercomplementary to the allelic sequence immediately 3 to the polymorphicsite is permitted to hybridize to a target molecule obtained from aparticular animal or human. If the polymorphic site on the targetmolecule contains a nucleotide that is complementary to the particularexonuclease-resistant nucleotide derivative present, then thatderivative will be incorporated onto the end of the hybridized primer.Such incorporation renders the primer resistant to exonuclease, andthereby permits its detection. Since the identity of theexonuclease-resistant derivative of the sample is known, a finding thatthe primer has become resistant to exonucleases reveals that thenucleotide(s) present in the polymorphic site of the target molecule iscomplementary to that of the nucleotide derivative used in the reaction.This method has the advantage that it does not require the determinationof large amounts of extraneous sequence data.

A solution-based method can be used for determining the identity of anucleotide of a polymorphic site. Cohen, D. et al. (French Patent2,650,840; PCT Appln. No. WO91/02087). As in the Mundy method of U.S.Pat. No. 4,656,127, a primer is employed that is complementary toallelic sequences immediately 3′ to a polymorphic site. The methoddetermines the identity of the nucleotide of that site using labeleddideoxynucleotide derivatives, which, if complementary to the nucleotideof the polymorphic site will become incorporated onto the terminus ofthe primer. An alternative method, known as Genetic Bit Analysis or GBAis described by Goelet, P. et al. (PCT Appln. No. 92/15712). The methodof Goelet, P. et al. uses mixtures of labeled terminators and a primerthat is complementary to the sequence 3′ to a polymorphic site. Thelabeled terminator that is incorporated is thus determined by, andcomplementary to, the nucleotide present in the polymorphic site of thetarget molecule being evaluated. In contrast to the method of Cohen etal. (French Patent 2,650,840; PCT Appln. No. WO91/02087) the method ofGoelet, P. et al. can be a heterogeneous phase assay, in which theprimer or the target molecule is immobilized to a solid phase.

Several primer-guided nucleotide incorporation procedures for assayingpolymorphic sites in DNA have been described (Komher, J. S. et al.,Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, B. P., Nucl. Acids Res.18:3671 (1990); Syvanen, A.-C., et al., Genomics 8:684-692 (1990);Kuppuswamy, M. N. et al., Proc. Nal. Acad. Sci. (U.S.A.) 88:1143-1147(1991); Prezant, T. R. et al., Hum. Mutat. 1:159-164 (1992); Ugozzoli,L. et al., GATA 9:107-112 (1992): Nyren, P. et al., Anal. Biochem.208:171-175 (1993)). These methods differ from GBA in that they utilizeincorporation of labeled deoxynucleotides to discriminate between basesat a polymorphic site. In such a format, since the signal isproportional to the number of deoxynucleotides incorporated,polymorphisms that occur in runs of the same nucleotide can result insignals that are proportional to the length of the run (Syvanen, A.-C.,et al., Amer. J. Hum. Genet. 52:46-59 (1993)).

A number of initiatives obtain sequence information directly frommillions of individual molecules of DNA or RNA in parallel. Real-timesingle molecule sequencing-by-synthesis technologies rely on thedetection of fluorescent nucleotides as they are incorporated into anascent strand of DNA that is complementary to the template beingsequenced. In one method, oligonucleotides 30-50 bases in length arecovalently anchored at the 5′ end to glass cover slips. These anchoredstrands perform two functions. First, they act as capture sites for thetarget template strands if the templates are configured with capturetails complementary to the surface-bound oligonucleotides. They also actas primers for the template directed primer extension that forms thebasis of the sequence reading. The capture primers function as a fixedposition site for sequence determination using multiple cycles ofsynthesis, detection, and chemical cleavage of the dye-linker to removethe dye. Each cycle consists of adding the polymerase/labeled nucleotidemixture, rinsing, imaging and cleavage of dye. In an alternative method,polymerase is modified with a fluorescent donor molecule and immobilizedon a glass slide, while each nucleotide is color-coded with an acceptorfluorescent moiety attached to a gamma-phosphate. The system detects theinteraction between a fluorescently-tagged polymerase and afluorescently modified nucleotide as the nucleotide becomes incorporatedinto the de novo chain. Other sequencing-by-synthesis technologies alsoexist.

Any suitable sequencing-by-synthesis platform can be used to identifymutations. As described above, four major sequencing-by-synthesisplatforms are currently available: the Genome Sequencers from Roche/454Life Sciences, the 1G Analyzer from Illumina/Solexa, the SOLiD systemfrom Applied BioSystems, and the Heliscope system from HelicosBiosciences. Sequencing-by-synthesis platforms have also been describedby Pacific BioSciences and VisiGen Biotechnologies. In some embodiments,a plurality of nucleic acid molecules being sequenced is bound to asupport (e.g., solid support). To immobilize the nucleic acid on asupport, a capture sequence/universal priming site can be added at the3′ and/or 5′ end of the template. The nucleic acids can be bound to thesupport by hybridizing the capture sequence to a complementary sequencecovalently attached to the support. The capture sequence (also referredto as a universal capture sequence) is a nucleic acid sequencecomplementary to a sequence attached to a support that may dually serveas a universal primer.

As an alternative to a capture sequence, a member of a coupling pair(such as, e.g., antibody/antigen, receptor/ligand, or the avidin-biotinpair as described in, e.g., US Patent Application No. 2006/0252077) canbe linked to each fragment to be captured on a surface coated with arespective second member of that coupling pair.

Subsequent to the capture, the sequence can be analyzed, for example, bysingle molecule detection/sequencing, e.g., as described in the Examplesand in U.S. Pat. No. 7,283,337, including template-dependentsequencing-by-synthesis. In sequencing-by-synthesis, the surface-boundmolecule is exposed to a plurality of labeled nucleotide triphosphatesin the presence of polymerase. The sequence of the template isdetermined by the order of labeled nucleotides incorporated into the 3′end of the growing chain. This can be done in real time or can be donein a step-and-repeat mode. For real-time analysis, different opticallabels to each nucleotide can be incorporated and multiple lasers can beutilized for stimulation of incorporated nucleotides.

Sequencing can also include other massively parallel sequencing or nextgeneration sequencing (NGS) techniques and platforms. Additionalexamples of massively parallel sequencing techniques and platforms arethe Illumina HiSeq or MiSeq, Thermo PGM or Proton, the Pac Bio RS II orSequel, Qiagen's Gene Reader, and the Oxford Nanopore MinION. Additionalsimilar current massively parallel sequencing technologies can be used,as well as future generations of these technologies.

Any cell type or tissue can be utilized to obtain nucleic acid samplesfor use in methods described herein. For example, a DNA or RNA samplecan be obtained from a tumor or a bodily fluid, e.g., blood, obtained byknown techniques (e.g. venipuncture) or saliva. Alternatively, nucleicacid tests can be performed on dry samples (e.g. hair or skin). Inaddition, a sample can be obtained for sequencing from a tumor andanother sample can be obtained from normal tissue for sequencing wherethe normal tissue is of the same tissue type as the tumor. A sample canbe obtained for sequencing from a tumor and another sample can beobtained from normal tissue for sequencing where the normal tissue is ofa distinct tissue type relative to the tumor.

Tumors can include one or more of lung cancer, melanoma, breast cancer,ovarian cancer, prostate cancer, kidney cancer, gastric cancer, coloncancer, testicular cancer, head and neck cancer, pancreatic cancer,brain cancer, B-cell lymphoma, acute myelogenous leukemia, chronicmyelogenous leukemia, chronic lymphocytic leukemia, and T-celllymphocytic leukemia, non-small cell lung cancer, and small cell lungcancer.

Alternatively, protein mass spectrometry can be used to identify orvalidate the presence of mutated peptides bound to MHC proteins on tumorcells. Peptides can be acid-eluted from tumor cells or from HLAmolecules that are immunoprecipitated from tumor, and then identifiedusing mass spectrometry.

IV. Neoantigens

Neoantigens can include nucleotides or polypeptides. For example, aneoantigen can be an RNA sequence that encodes for a polypeptidesequence. Neoantigens useful in vaccines can therefore includenucleotide sequences or polypeptide sequences.

Disclosed herein are isolated peptides that comprise tumor specificmutations identified by the methods disclosed herein, peptides thatcomprise known tumor specific mutations, and mutant polypeptides orfragments thereof identified by methods disclosed herein. Neoantigenpeptides can be described in the context of their coding sequence wherea neoantigen includes the nucleotide sequence (e.g., DNA or RNA) thatcodes for the related polypeptide sequence.

One or more polypeptides encoded by a neoantigen nucleotide sequence cancomprise at least one of: a binding affinity with MHC with an IC50 valueof less than 10 nM, for MHC Class I peptides a length of 8-15, 8, 9, 10,11, 12, 13, 14, or 15 amino acids, presence of sequence motifs within ornear the peptide promoting proteasome cleavage, and presence or sequencemotifs promoting TAP transport. For MHC Class I peptides a length 6-30,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 amino acids, presence of sequence motifswithin or near the peptide promoting cleavage by extracellular orlysosomal proteases (e.g., cathepsins) or HLA-DM catalyzed HLA binding.

One or more neoantigens can be presented on the surface of a tumor.

One or more neoantigens can be is immunogenic in a subject having atumor, e.g., capable of eliciting a T-cell response or a B cell responsein the subject.

One or more neoantigens that induce an autoimmune response in a subjectcan be excluded from consideration in the context of vaccine generationfor a subject having a tumor.

The size of at least one neoantigenic peptide molecule can comprise, butis not limited to, about 5, about 6, about 7, about 8, about 9, about10, about 11, about 12, about 13, about 14, about 15, about 16, about17, about 18, about 19, about 20, about 21, about 22, about 23, about24, about 25, about 26, about 27, about 28, about 29, about 30, about31, about 32, about 33, about 34, about 35, about 36, about 37, about38, about 39, about 40, about 41, about 42, about 43, about 44, about45, about 46, about 47, about 48, about 49, about 50, about 60, about70, about 80, about 90, about 100, about 110, about 120 or greater aminomolecule residues, and any range derivable therein. In specificembodiments the neoantigenic peptide molecules are equal to or less than50 amino acids.

Neoantigenic peptides and polypeptides can be: for MHC Class I 15residues or less in length and usually consist of between about 8 andabout 11 residues, particularly 9 or 10 residues: for MHC Class II, 6-30residues, inclusive.

If desirable, a longer peptide can be designed in several ways. In onecase, when presentation likelihoods of peptides on HLA alleles arepredicted or known, a longer peptide could consist of either: (1)individual presented peptides with an extensions of 2-5 amino acidstoward the N- and C-terminus of each corresponding gene product; (2) aconcatenation of some or all of the presented peptides with extendedsequences for each. In another case, when sequencing reveals a long (>10residues) neoepitope sequence present in the tumor (e.g. due to aframeshift, read-through or intron inclusion that leads to a novelpeptide sequence), a longer peptide would consist of (3) the entirestretch of novel tumor-specific amino acids-thus bypassing the need forcomputational or in vitro test-based selection of the strongestHLA-presented shorter peptide. In both cases, use of a longer peptideallows endogenous processing by patient-cells and may lead to moreeffective antigen presentation and induction of T-cell responses.

Neoantigenic peptides and polypeptides can be presented on an HLAprotein. In some aspects neoantigenic peptides and polypeptides arepresented on an HLA protein with greater affinity than a wild-typepeptide. In some aspects, a neoantigenic peptide or polypeptide can havean IC50 of at least less than 5000 nM, at least less than 1000 nM, atleast less than 500 nM, at least less than 250 nM, at least less than200 nM, at least less than 150 nM, at least less than 100 nM, at leastless than 50 nM or less.

In some aspects, neoantigenic peptides and polypeptides do not induce anautoimmune response and/or invoke immunological tolerance whenadministered to a subject.

Also provided are compositions comprising at least two or moreneoantigenic peptides. In some embodiments the composition contains atleast two distinct peptides. At least two distinct peptides can bederived from the same polypeptide. By distinct polypeptides is meantthat the peptide vary by length, amino acid sequence, or both. Thepeptides are derived from any polypeptide known to or have been found tocontain a tumor specific mutation. Suitable polypeptides from which theneoantigenic peptides can be derived can be found for example in theCOSMIC database. COSMIC curates comprehensive information on somaticmutations in human cancer. The peptide contains the tumor specificmutation. In some aspects the tumor specific mutation is a drivermutation for a particular cancer type.

Neoantigenic peptides and polypeptides having a desired activity orproperty can be modified to provide certain desired attributes, e.g.,improved pharmacological characteristics, while increasing or at leastretaining substantially all of the biological activity of the unmodifiedpeptide to bind the desired MHC molecule and activate the appropriateT-cell. For instance, neoantigenic peptide and polypeptides can besubject to various changes, such as substitutions, either conservativeor non-conservative, where such changes might provide for certainadvantages in their use, such as improved MHC binding, stability orpresentation. By conservative substitutions is meant replacing an aminoacid residue with another which is biologically and/or chemicallysimilar, e.g., one hydrophobic residue for another, or one polar residuefor another. The substitutions include combinations such as Gly, Ala;Val, Ile, Leu, Met; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe,Tyr. The effect of single amino acid substitutions may also be probedusing D-amino acids. Such modifications can be made using well knownpeptide synthesis procedures, as described in e.g., Merrifield, Science232:341-347 (1986), Barany & Merrifield, The Peptides, Gross &Meienhofer, eds. (N.Y., Academic Press), pp. 1-284 (1979); and Stewart &Young, Solid Phase Peptide Synthesis, (Rockford, Ill., Pierce), 2d Ed.(1984).

Modifications of peptides and polypeptides with various amino acidmimetics or unnatural amino acids can be particularly useful inincreasing the stability of the peptide and polypeptide in vivo.Stability can be assayed in a number of ways. For instance, peptidasesand various biological media, such as human plasma and serum, have beenused to test stability. See, e.g., Verhoef et al., Eur. J. Drug MetabPharmacokin. 11:291-302 (1986). Half-life of the peptides can beconveniently determined using a 25% human serum (vrv) assay. Theprotocol is generally as follows. Pooled human serum (Type AB, non-heatinactivated) is delipidated by centrifugation before use. The serum isthen diluted to 25% with RPMI tissue culture media and used to testpeptide stability. At predetermined time intervals a small amount ofreaction solution is removed and added to either 6% aqueoustrichloracetic acid or ethanol. The cloudy reaction sample is cooled (4degrees C.) for 15 minutes and then spun to pellet the precipitatedserum proteins. The presence of the peptides is then determined byreversed-phase HPLC using stability-specific chromatography conditions.

The peptides and polypeptides can be modified to provide desiredattributes other than improved serum half-life. For instance, theability of the peptides to induce CTL activity can be enhanced bylinkage to a sequence which contains at least one epitope that iscapable of inducing a T helper cell response. Immunogenic peptides-Thelper conjugates can be linked by a spacer molecule. The spacer istypically comprised of relatively small, neutral molecules, such asamino acids or amino acid mimetics, which are substantially unchargedunder physiological conditions. The spacers are typically selected from,e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids orneutral polar amino acids. It will be understood that the optionallypresent spacer need not be comprised of the same residues and thus canbe a hetero- or homo-oligomer. When present, the spacer will usually beat least one or two residues, more usually three to six residues.Alternatively, the peptide can be linked to the T helper peptide withouta spacer.

A neoantigenic peptide can be linked to the T helper peptide eitherdirectly or via a spacer either at the amino or carboxy terminus of thepeptide. The amino terminus of either the neoantigenic peptide or the Thelper peptide can be acylated. Exemplary T helper peptides includetetanus toxoid 830-843, influenza 307-319, malaria circumsporozoite382-398 and 378-389.

Proteins or peptides can be made by any technique known to those ofskill in the art, including the expression of proteins, polypeptides orpeptides through standard molecular biological techniques, the isolationof proteins or peptides from natural sources, or the chemical synthesisof proteins or peptides. The nucleotide and protein, polypeptide andpeptide sequences corresponding to various genes have been previouslydisclosed, and can be found at computerized databases known to those ofordinary skill in the art. One such database is the National Center forBiotechnology Information's Genbank and GenPept databases located at theNational Institutes of Health website. The coding regions for knowngenes can be amplified and/or expressed using the techniques disclosedherein or as would be known to those of ordinary skill in the art.Alternatively, various commercial preparations of proteins, polypeptidesand peptides are known to those of skill in the art.

In a further aspect a neoantigen includes a nucleic acid (e.g.polynucleotide) that encodes a neoantigenic peptide or portion thereof.The polynucleotide can be, e.g., DNA, cDNA, PNA, CNA, RNA (e.g., mRNA),either single- and/or double-stranded, or native or stabilized forms ofpolynucleotides, such as, e.g., polynucleotides with a phosphorothiatebackbone, or combinations thereof and it may or may not contain introns.A still further aspect provides an expression vector capable ofexpressing a polypeptide or portion thereof. Expression vectors fordifferent-cell types are well known in the art and can be selectedwithout undue experimentation. Generally, DNA is inserted into anexpression vector, such as a plasmid, in proper orientation and correctreading frame for expression. If necessary, DNA can be linked to theappropriate transcriptional and translational regulatory controlnucleotide sequences recognized by the desired host, although suchcontrols are generally available in the expression vector. The vector isthen introduced into the host through standard techniques. Guidance canbe found e.g. in Sambrook et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

IV. Vaccine Compositions

Also disclosed herein is an immunogenic composition, e.g., a vaccinecomposition, capable of raising a specific immune response, e.g., atumor-specific immune response. Vaccine compositions typically comprisea plurality of neoantigens, e.g., selected using a method describedherein. Vaccine compositions can also be referred to as vaccines.

A vaccine can contain between 1 and 30 peptides, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 different peptides, 6, 7, 8, 9, 10 11, 12, 13, or 14different peptides, or 12, 13 or 14 different peptides. Peptides caninclude post-translational modifications. A vaccine can contain between1 and 100 or more nucleotide sequences, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100 ormore different nucleotide sequences, 6, 7, 8, 9, 10 11, 12, 13, or 14different nucleotide sequences, or 12, 13 or 14 different nucleotidesequences. A vaccine can contain between 1 and 30 neoantigen sequences,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100 or more different neoantigen sequences, 6,7, 8, 9, 10 11, 12, 13, or 14 different neoantigen sequences, or 12, 13or 14 different neoantigen sequences.

In one embodiment, different peptides and/or polypeptides or nucleotidesequences encoding them are selected so that the peptides and/orpolypeptides capable of associating with different MHC molecules, suchas different MHC class I molecules and/or different MHC class IImolecules. In some aspects, one vaccine composition comprises codingsequence for peptides and/or polypeptides capable of associating withthe most frequently occurring MHC class I molecules and/or MHC class IImolecules. Hence, vaccine compositions can comprise different fragmentscapable of associating with at least 2 preferred, at least 3 preferred,or at least 4 preferred MHC class I molecules and/or MHC class IImolecules.

The vaccine composition can be capable of raising a specific cytotoxicT-cells response and/or a specific helper T-cell response.

A vaccine composition can further comprise an adjuvant and/or a carrier.Examples of useful adjuvants and carriers are given herein below. Acomposition can be associated with a carrier such as e.g. a protein oran antigen-presenting cell such as e.g. a dendritic cell (DC) capable ofpresenting the peptide to a T-cell.

Adjuvants are any substance whose admixture into a vaccine compositionincreases or otherwise modifies the immune response to a neoantigen.Carriers can be scaffold structures, for example a polypeptide or apolysaccharide, to which a neoantigen, is capable of being associated.Optionally, adjuvants are conjugated covalently or non-covalently.

The ability of an adjuvant to increase an immune response to an antigenis typically manifested by a significant or substantial increase in animmune-mediated reaction, or reduction in disease symptoms. For example,an increase in humoral immunity is typically manifested by a significantincrease in the titer of antibodies raised to the antigen, and anincrease in T-cell activity is typically manifested in increased cellproliferation, or cellular cytotoxicity, or cytokine secretion. Anadjuvant may also alter an immune response, for example, by changing aprimarily humoral or Th response into a primarily cellular, or Thresponse.

Suitable adjuvants include, but are not limited to 1018 ISS, alum,aluminium salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM,GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS,ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, MontanideIMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51,OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel vector system, PLGmicroparticles, resiquimod, SRL172, Virosomes and other Virus-likeparticles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21stimulon (Aquila Biotech, Worcester, Mass., USA) which is derived fromsaponin, mycobacterial extracts and synthetic bacterial cell wallmimics, and other proprietary adjuvants such as Ribi's Detox. Quil orSuperfos. Adjuvants such as incomplete Freund's or GM-CSF are useful.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Dupuis M, etal., Cell Immunol. 1998; 186(1):18-27; Allison A C: Dev Biol Stand. 199892:3-11). Also cytokines can be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-alpha), accelerating the maturation of dendriticcells into efficient antigen-presenting cells for T-lymphocytes (e.g.,GM-CSF, IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specificallyincorporated herein by reference in its entirety) and acting asimmunoadjuvants (e.g., IL-12) (Gabrilovich D I, et al., J ImmunotherEmphasis Tumor Immunol. 1996 (6):414-418).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Other TLR bindingmolecules such as RNA binding TLR 7, TLR 8 and/or TLR 9 may also beused.

Other examples of useful adjuvants include, but are not limited to,chemically modified CpGs (e.g. CpR, Idera), Poly(I:C)(e.g. polyi:CI2U),non-CpG bacterial DNA or RNA as well as immunoactive small molecules andantibodies such as cyclophosphamide, sunitinib, bevacizumab, celebrex,NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999,CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, andSC58175, which may act therapeutically and/or as an adjuvant. Theamounts and concentrations of adjuvants and additives can readily bedetermined by the skilled artisan without undue experimentation.Additional adjuvants include colony-stimulating factors, such asGranulocyte Macrophage Colony Stimulating Factor (GM-CSF, sargramostim).

A vaccine composition can comprise more than one different adjuvant.Furthermore, a therapeutic composition can comprise any adjuvantsubstance including any of the above or combinations thereof. It is alsocontemplated that a vaccine and an adjuvant can be administered togetheror separately in any appropriate sequence.

A carrier (or excipient) can be present independently of an adjuvant.The function of a carrier can for example be to increase the molecularweight of in particular mutant to increase activity or immunogenicity,to confer stability, to increase the biological activity, or to increaseserum half-life. Furthermore, a carrier can aid presenting peptides toT-cells. A carrier can be any suitable carrier known to the personskilled in the art, for example a protein or an antigen presenting cell.A carrier protein could be but is not limited to keyhole limpethemocyanin, serum proteins such as transferrin, bovine serum albumin,human serum albumin. thyroglobulin or ovalbumin, immunoglobulins, orhormones, such as insulin or palmitic acid. For immunization of humans,the carrier is generally a physiologically acceptable carrier acceptableto humans and safe. However, tetanus toxoid and/or diptheria toxoid aresuitable carriers. Alternatively, the carrier can be dextrans forexample sepharose.

Cytotoxic T-cells (CTLs) recognize an antigen in the form of a peptidebound to an MHC molecule rather than the intact foreign antigen itself.The MHC molecule itself is located at the cell surface of an antigenpresenting cell. Thus, an activation of CTLs is possible if a trimericcomplex of peptide antigen, MHC molecule, and APC is present.Correspondingly, it may enhance the immune response if not only thepeptide is used for activation of CTLs, but if additionally APCs withthe respective MHC molecule are added. Therefore, in some embodiments avaccine composition additionally contains at least one antigenpresenting cell.

Neoantigens can also be included in viral vector-based vaccineplatforms, such as vaccinia, fowlpox, self-replicating alphavirus,marabavirus, adenovirus (See, e.g., Tatsis et al., Adenoviruses,Molecular Therapy (2004) 10, 616-629), or lentivirus, including but notlimited to second, third or hybrid second/third generation lentivirusand recombinant lentivirus of any generation designed to target specificcell types or receptors (See, e.g., Hu et al., Immunization Delivered byLentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev.(2011) 239(1): 45-61, Sakuma et al., Lentiviral vectors: basic totranslational, Biochem J. (2012) 443(3):603-18, Cooper et al., Rescue ofsplicing-mediated intron loss maximizes expression in lentiviral vectorscontaining the human ubiquitin C promoter, Nucl. Acids Res. (2015) 43(1): 682-690, Zufferey et al., Self-Inactivating Lentivirus Vector forSafe and Efficient In Vivo Gene Delivery, J. Virol. (1998) 72 (12):9873-9880). Dependent on the packaging capacity of the above mentionedviral vector-based vaccine platforms, this approach can deliver one ormore nucleotide sequences that encode one or more neoantigen peptides.The sequences may be flanked by non-mutated sequences, may be separatedby linkers or may be preceded with one or more sequences targeting asubcellular compartment (See, e.g., Gros et al., Prospectiveidentification of neoantigen-specific lymphocytes in the peripheralblood of melanoma patients, Nat Med. (2016) 22 (4):433-8, Stronen etal., Targeting of cancer neoantigens with donor-derived T-cell receptorrepertoires, Science. (2016) 352 (6291):1337-41, Lu et al., Efficientidentification of mutated cancer antigens recognized by T-cellsassociated with durable tumor regressions, Clin Cancer Res. (2014)20(13):3401-10). Upon introduction into a host, infected cells expressthe neoantigens, and thereby elicit a host immune (e.g., CTL) responseagainst the peptide(s). Vaccinia vectors and methods useful inimmunization protocols are described in, e.g., U.S. Pat. No. 4,722,848.Another vector is BCG (Bacille Calmette Guerin). BCG vectors aredescribed in Stover et al. (Nature 351:456-460 (1991)). A wide varietyof other vaccine vectors useful for therapeutic administration orimmunization of neoantigens, e.g., Salmonella typhi vectors, and thelike will be apparent to those skilled in the art from the descriptionherein.

IV.A. Additional Considerations for Vaccine Design and ManufactureIV.A.1. Determination of a Set of Peptides that Cover all TumorSubclones

Truncal peptides, meaning those presented by all or most tumorsubclones, will be prioritized for inclusion into the vaccine.⁵³Optionally, if there are no truncal peptides predicted to be presentedand immunogenic with high probability, or if the number of truncalpeptides predicted to be presented and immunogenic with high probabilityis small enough that additional non-truncal peptides can be included inthe vaccine, then further peptides can be prioritized by estimating thenumber and identity of tumor subclones and choosing peptides so as tomaximize the number of tumor subclones covered by the vaccine.

IV.A.2. Neoantigen Prioritization

After all of the above neoantigen filters are applied, more candidateneoantigens may still be available for vaccine inclusion than thevaccine technology can support. Additionally, uncertainty about variousaspects of the neoantigen analysis may remain and tradeoffs may existbetween different properties of candidate vaccine neoantigens. Thus, inplace of predetermined filters at each step of the selection process, anintegrated multi-dimensional model can be considered that placescandidate neoantigens in a space with at least the following axes andoptimizes selection using an integrative approach.

-   -   1. Risk of auto-immunity or tolerance (risk of germline) (lower        risk of auto-immunity is typically preferred)    -   2. Probability of sequencing artifact (lower probability of        artifact is typically preferred)    -   3. Probability of immunogenicity (higher probability of        immunogenicity is typically preferred)    -   4. Probability of presentation (higher probability of        presentation is typically preferred)    -   5. Gene expression (higher expression is typically preferred)    -   6. Coverage of HLA genes (larger number of HLA molecules        involved in the presentation of a set of neoantigens may lower        the probability that a tumor will escape immune attack via        downregulation or mutation of HLA molecules)    -   7. Coverage of HLA classes (covering both HLA-I and HLA-II may        increase the probability of therapeutic response and decrease        the probability of tumor escape)

V. Therapeutic and Manufacturing Methods

Also provided is a method of inducing a tumor specific immune responsein a subject, vaccinating against a tumor, treating and or alleviating asymptom of cancer in a subject by administering to the subject one ormore neoantigens such as a plurality of neoantigens identified usingmethods disclosed herein.

In some aspects, a subject has been diagnosed with cancer or is at riskof developing cancer. A subject can be a human, dog, cat, horse or anyanimal in which a tumor specific immune response is desired. A tumor canbe any solid tumor such as breast, ovarian, prostate, lung, kidney,gastric, colon, testicular, head and neck, pancreas, brain, melanoma,and other tumors of tissue organs and hematological tumors, such aslymphomas and leukemias, including acute myelogenous leukemia, chronicmyelogenous leukemia, chronic lymphocytic leukemia, T-cell lymphocyticleukemia, and B cell lymphomas.

A neoantigen can be administered in an amount sufficient to induce a CTLresponse.

A neoantigen can be administered alone or in combination with othertherapeutic agents. The therapeutic agent is for example, achemotherapeutic agent, radiation, or immunotherapy. Any suitabletherapeutic treatment for a particular cancer can be administered.

In addition, a subject can be further administered ananti-immunosuppressive/immunostimulatory agent such as a checkpointinhibitor. For example, the subject can be further administered ananti-CTLA antibody or anti-PD-1 or anti-PD-L1. Blockade of CTLA-4 orPD-L1 by antibodies can enhance the immune response to cancerous cellsin the patient. In particular, CTLA-4 blockade has been shown effectivewhen following a vaccination protocol.

The optimum amount of each neoantigen to be included in a vaccinecomposition and the optimum dosing regimen can be determined. Forexample, a neoantigen or its variant can be prepared for intravenous(i.v.) injection, sub-cutaneous (s.c.) injection, intradermal (i.d.)injection, intraperitoneal (i.p.) injection, intramuscular (i.m.)injection. Methods of injection include s.c., i.d., i.p., i.m., and i.v.Methods of DNA or RNA injection include i.d., i.m., s.c., i.p. and i.v.Other methods of administration of the vaccine composition are known tothose skilled in the art.

A vaccine can be compiled so that the selection, number and/or amount ofneoantigens present in the composition is/are tissue, cancer, and/orpatient-specific. For instance, the exact selection of peptides can beguided by expression patterns of the parent proteins in a given tissue.The selection can be dependent on the specific type of cancer, thestatus of the disease, earlier treatment regimens, the immune status ofthe patient, and, of course, the HLA-haplotype of the patient.Furthermore, a vaccine can contain individualized components, accordingto personal needs of the particular patient. Examples include varyingthe selection of neoantigens according to the expression of theneoantigen in the particular patient or adjustments for secondarytreatments following a first round or scheme of treatment.

For a composition to be used as a vaccine for cancer, neoantigens withsimilar normal self-peptides that are expressed in high amounts innormal tissues can be avoided or be present in low amounts in acomposition described herein. On the other hand, if it is known that thetumor of a patient expresses high amounts of a certain neoantigen, therespective pharmaceutical composition for treatment of this cancer canbe present in high amounts and/or more than one neoantigen specific forthis particularly neoantigen or pathway of this neoantigen can beincluded.

Compositions comprising a neoantigen can be administered to anindividual already suffering from cancer. In therapeutic applications,compositions are administered to a patient in an amount sufficient toelicit an effective CTL response to the tumor antigen and to cure or atleast partially arrest symptoms and/or complications. An amount adequateto accomplish this is defined as “therapeutically effective dose.”Amounts effective for this use will depend on, e.g., the composition,the manner of administration, the stage and severity of the diseasebeing treated, the weight and general state of health of the patient,and the judgment of the prescribing physician. It should be kept in mindthat compositions can generally be employed in serious disease states,that is, life-threatening or potentially life threatening situations,especially when the cancer has metastasized. In such cases, in view ofthe minimization of extraneous substances and the relative nontoxicnature of a neoantigen, it is possible and can be felt desirable by thetreating physician to administer substantial excesses of thesecompositions.

For therapeutic use, administration can begin at the detection orsurgical removal of tumors. This is followed by boosting doses until atleast symptoms are substantially abated and for a period thereafter.

The pharmaceutical compositions (e.g., vaccine compositions) fortherapeutic treatment are intended for parenteral, topical, nasal, oralor local administration. A pharmaceutical compositions can beadministered parenterally, e.g., intravenously, subcutaneously,intradermally, or intramuscularly. The compositions can be administeredat the site of surgical excision to induce a local immune response tothe tumor. Disclosed herein are compositions for parenteraladministration which comprise a solution of the neoantigen and vaccinecompositions are dissolved or suspended in an acceptable carrier, e.g.,an aqueous carrier. A variety of aqueous carriers can be used, e.g.,water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid andthe like. These compositions can be sterilized by conventional, wellknown sterilization techniques, or can be sterile filtered. Theresulting aqueous solutions can be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterilesolution prior to administration. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc.

Neoantigens can also be administered via liposomes, which target them toa particular cells tissue, such as lymphoid tissue. Liposomes are alsouseful in increasing half-life. Liposomes include emulsions, foams,micelles, insoluble monolayers, liquid crystals, phospholipiddispersions, lamellar layers and the like. In these preparations theneoantigen to be delivered is incorporated as part of a liposome, aloneor in conjunction with a molecule which binds to, e.g., a receptorprevalent among lymphoid cells, such as monoclonal antibodies which bindto the CD45 antigen, or with other therapeutic or immunogeniccompositions. Thus, liposomes filled with a desired neoantigen can bedirected to the site of lymphoid cells, where the liposomes then deliverthe selected therapeutic/immunogenic compositions. Liposomes can beformed from standard vesicle-forming lipids, which generally includeneutral and negatively charged phospholipids and a sterol, such ascholesterol. The selection of lipids is generally guided byconsideration of, e.g., liposome size, acid lability and stability ofthe liposomes in the blood stream. A variety of methods are availablefor preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev.Biophys. Bioeng. 9; 467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728,4,501,728, 4,837,028, and 5,019,369.

For targeting to the immune cells, a ligand to be incorporated into theliposome can include, e.g., antibodies or fragments thereof specific forcell surface determinants of the desired immune system cells. A liposomesuspension can be administered intravenously, locally, topically, etc.in a dose which varies according to, inter alia, the manner ofadministration, the peptide being delivered, and the stage of thedisease being treated.

For therapeutic or immunization purposes, nucleic acids encoding apeptide and optionally one or more of the peptides described herein canalso be administered to the patient. A number of methods areconveniently used to deliver the nucleic acids to the patient. Forinstance, the nucleic acid can be delivered directly, as “naked DNA”.This approach is described, for instance, in Wolff et al., Science 247:1465-1468 (1990) as well as U.S. Pat. Nos. 5,580,859 and 5,589,466. Thenucleic acids can also be administered using ballistic delivery asdescribed, for instance, in U.S. Pat. No. 5,204,253. Particles comprisedsolely of DNA can be administered. Alternatively, DNA can be adhered toparticles, such as gold particles. Approaches for delivering nucleicacid sequences can include viral vectors, mRNA vectors, and DNA vectorswith or without electroporation.

The nucleic acids can also be delivered complexed to cationic compounds,such as cationic lipids. Lipid-mediated gene delivery methods aredescribed, for instance, in 9618372WOAWO 96/18372: 9324640WOAWO93/24640: Mannino & Gould-Fogerite, BioTechniques 6(7): 682-691 (1988);U.S. Pat. No. 5,279,833 Rose U.S. Pat. No. 5,279,833; 9106309WOAWO91/06309; and Felgner et al., Proc. Natil. Acad. Sci. USA 84: 7413-7414(1987).

Neoantigens can also be included in viral vector-based vaccineplatforms, such as vaccinia, fowlpox, self-replicating alphavirus,marabavirus, adenovirus (See. e.g., Tatsis et al., Adenoviruses,Molecular Therapy (2004) 10, 616-629), or lentivirus, including but notlimited to second, third or hybrid second/third generation lentivirusand recombinant lentivirus of any generation designed to target specificcell types or receptors (See, e.g., Hu et al., Immunization Delivered byLentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev.(2011) 239(1): 45-61, Sakuma et al., Lentiviral vectors: basic totranslational, Biochem J. (2012) 443(3):603-18, Cooper et al., Rescue ofsplicing-mediated intron loss maximizes expression in lentiviral vectorscontaining the human ubiquitin C promoter, Nucl. Acids Res. (2015) 43(1): 682-690, Zufferey et al., Self-Inactivating Lentivirus Vector forSafe and Efficient In Vivo Gene Delivery, J. Virol. (1998) 72 (12):9873-9880). Dependent on the packaging capacity of the above mentionedviral vector-based vaccine platforms, this approach can deliver one ormore nucleotide sequences that encode one or more neoantigen peptides.The sequences may be flanked by non-mutated sequences, may be separatedby linkers or may be preceded with one or more sequences targeting asubcellular compartment (See, e.g., Gros et al., Prospectiveidentification of neoantigen-specific lymphocytes in the peripheralblood of melanoma patients, Nat Med. (2016) 22 (4):433-8, Stronen etal., Targeting of cancer neoantigens with donor-derived T-cell receptorrepertoires, Science. (2016) 352 (6291):1337-41. Lu et al., Efficientidentification of mutated cancer antigens recognized by T-cellsassociated with durable tumor regressions, Clin Cancer Res. (2014)20(13):3401-10). Upon introduction into a host, infected cells expressthe neoantigens, and thereby elicit a host immune (e.g., CTL) responseagainst the peptide(s). Vaccinia vectors and methods useful inimmunization protocols are described in, e.g., U.S. Pat. No. 4,722,848.Another vector is BCG (Bacille Calmette Guerin). BCG vectors aredescribed in Stover et al. (Nature 351:456-460 (1991)). A wide varietyof other vaccine vectors useful for therapeutic administration orimmunization of neoantigens, e.g., Salmonella typhi vectors, and thelike will be apparent to those skilled in the art from the descriptionherein.

A means of administering nucleic acids uses minigene constructs encodingone or multiple epitopes. To create a DNA sequence encoding the selectedCTL epitopes (minigene) for expression in human cells, the amino acidsequences of the epitopes are reverse translated. A human codon usagetable is used to guide the codon choice for each amino acid. Theseepitope-encoding DNA sequences are directly adjoined, creating acontinuous polypeptide sequence. To optimize expression and/orimmunogenicity, additional elements can be incorporated into theminigene design. Examples of amino acid sequence that could be reversetranslated and included in the minigene sequence include: helper Tlymphocyte, epitopes, a leader (signal) sequence, and an endoplasmicreticulum retention signal. In addition, MHC presentation of CTLepitopes can be improved by including synthetic (e.g. poly-alanine) ornaturally-occurring flanking sequences adjacent to the CTL epitopes. Theminigene sequence is converted to DNA by assembling oligonucleotidesthat encode the plus and minus strands of the minigene. Overlappingoligonucleotides (30-100 bases long) are synthesized, phosphorylated,purified and annealed under appropriate conditions using well knowntechniques. The ends of the oligonucleotides are joined using T4 DNAligase. This synthetic minigene, encoding the CTL epitope polypeptide,can then cloned into a desired expression vector.

Purified plasmid DNA can be prepared for injection using a variety offormulations. The simplest of these is reconstitution of lyophilized DNAin sterile phosphate-buffer saline (PBS). A variety of methods have beendescribed, and new techniques can become available. As noted above,nucleic acids are conveniently formulated with cationic lipids. Inaddition, glycolipids, fusogenic liposomes, peptides and compoundsreferred to collectively as protective, interactive, non-condensing(PINC) could also be complexed to purified plasmid DNA to influencevariables such as stability, intramuscular dispersion, or trafficking tospecific organs or cell types.

Also disclosed is a method of manufacturing a tumor vaccine, comprisingperforming the steps of a method disclosed herein; and producing a tumorvaccine comprising a plurality of neoantigens or a subset of theplurality of neoantigens.

Neoantigens disclosed herein can be manufactured using methods known inthe art. For example, a method of producing a neoantigen or a vector(e.g., a vector including at least one sequence encoding one or moreneoantigens) disclosed herein can include culturing a host cell underconditions suitable for expressing the neoantigen or vector wherein thehost cell comprises at least one polynucleotide encoding the neoantigenor vector, and purifying the neoantigen or vector. Standard purificationmethods include chromatographic techniques, electrophoretic,immunological, precipitation, dialysis, filtration, concentration, andchromatofocusing techniques.

Host cells can include a Chinese Hamster Ovary (CHO) cell, NS0 cell,yeast, or a HEK293 cell. Host cells can be transformed with one or morepolynucleotides comprising at least one nucleic acid sequence thatencodes a neoantigen or vector disclosed herein, optionally wherein theisolated polynucleotide further comprises a promoter sequence operablylinked to the at least one nucleic acid sequence that encodes theneoantigen or vector. In certain embodiments the isolated polynucleotidecan be cDNA.

VI. Neoantigen Identification VIA. Neoantigen Candidate Identification

Research methods for NGS analysis of tumor and normal exome andtranscriptomes have been described and applied in the neoantigenidentification space.^(6,14-15) The example below considers certainoptimizations for greater sensitivity and specificity for neoantigenidentification in the clinical setting. These optimizations can begrouped into two areas, those related to laboratory processes and thoserelated to the NGS data analysis.

VI.A.1. Laboratory Process Optimizations

The process improvements presented here address challenges inhigh-accuracy neoantigen discovery from clinical specimens with lowtumor content and small volumes by extending concepts developed forreliable cancer driver gene assessment in targeted cancer panels¹⁶ tothe whole—exome and—transcriptome setting necessary for neoantigenidentification. Specifically, these improvements include:

-   -   1. Targeting deep (>500×) unique average coverage across the        tumor exome to detect mutations present at low mutant allele        frequency due to either low tumor content or subclonal state.    -   2. Targeting uniform coverage across the tumor exome, with <5%        of bases covered at <100×, so that the fewest possible        neoantigens are missed, by, for instance:        -   a. Employing DNA-based capture probes with individual probe            QC¹⁷        -   b. Including additional baits for poorly covered regions    -   3. Targeting uniform coverage across the normal exome, where <5%        of bases are covered at <20× so that the fewest neoantigens        possible remain unclassified for somatic/germline status (and        thus not usable as TSNAs)    -   4. To minimize the total amount of sequencing required, sequence        capture probes will be designed for coding regions of genes        only, as non-coding RNA cannot give rise to neoantigens.        Additional optimizations include:        -   a. supplementary probes for HLA genes, which are GC-rich and            poorly captured by standard exome sequencing¹⁸        -   b. exclusion of genes predicted to generate few or no            candidate neoantigens, due to factors such as insufficient            expression, suboptimal digestion by the proteasome, or            unusual sequence features.    -   5. Tumor RNA will likewise be sequenced at high depth (>100M        reads) in order to enable variant detection, quantification of        gene and splice-variant (“isoform”) expression, and fusion        detection. RNA from FFPE samples will be extracted using        probe-based enrichment, with the same or similar probes used to        capture exomes in DNA.

VI.A.2. NGS Data Analysis Optimizations

Improvements in analysis methods address the suboptimal sensitivity andspecificity of common research mutation calling approaches, andspecifically consider customizations relevant for neoantigenidentification in the clinical setting. These include:

-   -   1. Using the HG38 reference human genome or a later version for        alignment, as it contains multiple MHC regions assemblies better        reflective of population polymorphism, in contrast to previous        genome releases.    -   2. Overcoming the limitations of single variant callers²⁰ by        merging results from different programs⁵        -   a. Single-nucleotide variants and indels will be detected            from tumor DNA, tumor RNA and normal DNA with a suite of            tools including: programs based on comparisons of tumor and            normal DNA, such as Strelka²¹ and Mutect²² and programs that            incorporate tumor DNA, tumor RNA and normal DNA, such as            UNCeqR, which is particularly advantageous in low-purity            samples²³        -   b. Indels will be determined with programs that perform            local re-assembly, such as Strelka and ABRA²⁴        -   c. Structural rearrangements will be determined using            dedicated tools such as Pindel²⁵ or Breakseq²⁶.    -   3. In order to detect and prevent sample swaps, variant calls        from samples for the same patient will be compared at a chosen        number of polymorphic sites.    -   4. Extensive filtering of artefactual calls will be performed,        for instance, by:        -   a. Removal of variants found in normal DNA, potentially with            relaxed detection parameters in cases of low coverage, and            with a permissive proximity criterion in case of indels        -   b. Removal of variants due to low mapping quality or low            base quality²⁷.        -   c. Removal of variants stemming from recurrent sequencing            artifacts, even if not observed in the corresponding            normal²⁷. Examples include variants primarily detected on            one strand.        -   d. Removal of variants detected in an unrelated set of            controls²⁷    -   5. Accurate HLA calling from normal exome using one of        seq2HLA²⁸, ATHLATES²⁹ or Optitype and also combining exome and        RNA sequencing data²⁸. Additional potential optimizations        include the adoption of a dedicated assay for HLA typing such as        long-read DNA sequencing³⁰, or the adaptation of a method for        joining RNA fragments to retain continuity³¹.    -   6. Robust detection of neo-ORFs arising from tumor-specific        splice variants will be performed by assembling transcripts from        RNA-seq data using CLASS³², Bayesembler³³, StringTie³⁴ or a        similar program in its reference-guided mode (i.e., using known        transcript structures rather than attempting to recreate        transcripts in their entirety from each experiment). While        Cufflinks “is commonly used for this purpose, it frequently        produces implausibly large numbers of splice variants, many of        them far shorter than the full-length gene, and can fail to        recover simple positive controls. Coding sequences and        nonsense-mediated decay potential will be determined with tools        such as SpliceR³⁶ and MAMBA³⁷, with mutant sequences        re-introduced. Gene expression will be determined with a tool        such as Cufflinks” or Express (Roberts and Pachter, 2013).        Wild-type and mutant-specific expression counts and/or relative        levels will be determined with tools developed for these        purposes, such as ASE³⁸ or HTSeq³⁹. Potential filtering steps        include:        -   a. Removal of candidate neo-ORFs deemed to be insufficiently            expressed.        -   b. Removal of candidate neo-ORFs predicted to trigger            non-sense mediated decay (NMD).    -   7. Candidate neoantigens observed only in RNA (e.g., neoORFs)        that cannot directly be verified as tumor-specific will be        categorized as likely tumor-specific according to additional        parameters, for instance by considering:        -   a. Presence of supporting tumor DNA-only cis-acting            frameshift or splice-site mutations        -   b. Presence of corroborating tumor DNA-only trans-acting            mutation in a splicing factor. For instance, in three            independently published experiments with R625-mutant SF3B1,            the genes exhibiting the most differentially splicing were            concordant even though one experiment examined uveal            melanoma patients⁴⁰, the second a uveal melanoma cell            line⁴¹, and the third breast cancer patients⁴².        -   c. For novel splicing isoforms, presence of corroborating            “novel” splice-junction reads in the RNASeq data.        -   d. For novel re-arrangements, presence of corroborating            juxta-exon reads in tumor DNA that are absent from normal            DNA        -   e. Absence from gene expression compendium such as GTEx⁴³            (i.e. making germline origin less likely)    -   8. Complementing the reference genome alignment-based analysis        by comparing assembled DNA tumor and normal reads (or k-mers        from such reads) directly to avoid alignment and annotation        based errors and artifacts. (e.g. for somatic variants arising        near germline variants or repeat-context indels)

In samples with poly-adenylated RNA, the presence of viral and microbialRNA in the RNA-seq data will be assessed using RNA CoMPASS⁴⁴ or asimilar method, toward the identification of additional factors that maypredict patient response.

VI.B. Isolation and Detection of HLA Peptides

Isolation of HLA-peptide molecules was performed using classicimmunoprecipitation (IP) methods after lysis and solubilization of thetissue sample⁵⁵⁻⁵⁸. A clarified lysate was used for HLA specific IP.

Immunoprecipitation was performed using antibodies coupled to beadswhere the antibody is specific for HLA molecules. For a pan-Class I HLAimmunoprecipitation, a pan-Class I CR antibody is used, for Class IIHLA-DR, an HLA-DR antibody is used. Antibody is covalently attached toNHS-sepharose beads during overnight incubation. After covalentattachment, the beads were washed and aliquoted for IP.^(59, 60)Immunoprecipitations can also be performed with antibodies that are notcovalently attached to beads. Typically this is done using sepharose ormagnetic beads coated with Protein A and/or Protein G to hold theantibody to the column. Some antibodies that can be used to selectivelyenrich MHCpeptide complex are listed below.

Antibody Name Specificity W6/32 Class I HLA-A, B, C L243 Class II -HLA-DR Tu36 Class II - HLA-DR LN3 Class II - HLA-DR Tu39 Class II -HLA-DR, DP, DQ

The clarified tissue lysate is added to the antibody beads for theimmunoprecipitation. After immunoprecipitation, the beads are removedfrom the lysate and the lysate stored for additional experiments,including additional IPs. The IP beads are washed to remove non-specificbinding and the HLA/peptide complex is eluted from the beads usingstandard techniques. The protein components are removed from thepeptides using a molecular weight spin column or C18 fractionation. Theresultant peptides are taken to dryness by SpeedVac evaporation and insome instances are stored at −20 C prior to MS analysis.

Dried peptides are reconstituted in an HPLC buffer suitable for reversephase chromatography and loaded onto a C-18 microcapillary HPLC columnfor gradient elution in a Fusion Lumos mass spectrometer (Thermo). MS1spectra of peptide mass/charge (m/z) were collected in the Orbitrapdetector at high resolution followed by MS2 low resolution scanscollected in the ion trap detector after HCD fragmentation of theselected ion. Additionally, MS2 spectra can be obtained using either CIDor ETD fragmentation methods or any combination of the three techniquesto attain greater amino acid coverage of the peptide. MS2 spectra canalso be measured with high resolution mass accuracy in the Orbitrapdetector. MS2 spectra from each analysis are searched against a proteindatabase using Comet^(61, 62) and the peptide identification are scoredusing Percolator⁶³⁻⁶⁵. Additional sequencing is performed using PEAKSstudio (Bioinformatics Solutions Inc.) and other search engines orsequencing methods can be used including spectral matching and de novosequencing⁷⁵.

VI.B.1. MS Limit of Detection Studies in Support of Comprehensive HLAPeptide Sequencing

Using the peptide YVYVADVAAK (SEQ ID NO: 1) it was determined what thelimits of detection are using different amounts of peptide loaded ontothe LC column. The amounts of peptide tested were 1 pmol, 100 fmol, 10fmol, 1 fmol, and 100 amol. (Table 1) The results are shown in FIG. 1F.These results indicate that the lowest limit of detection (LoD) is inthe attomol range (10⁻¹⁸), that the dynamic range spans five orders ofmagnitude, and that the signal to noise appears sufficient forsequencing at low femtomol ranges (10⁻¹).

Peptide m/z Loaded on Column Copies/Cell in 1e9cells 566.830 1 pmol 600562.823 100 fmol 60 559.816 10 fmol 6 556.810 1 fmol 0.6 553.802 100amol 0.06

VI. Presentation Model VII.A. System Overview

FIG. 2A is an overview of an environment 100 for identifying likelihoodsof peptide presentation in patients, in accordance with an embodiment.The environment 100 provides context in order to introduce apresentation identification system 160, itself including a presentationinformation store 165.

The presentation identification system 160 is one or computer models,embodied in a computing system as discussed below with respect to FIG.30, that receives peptide sequences associated with a set of MHC allelesand determines likelihoods that the peptide sequences will be presentedby one or more of the set of associated MHC alleles. The presentationidentification system 160 may be applied to both class I and class IIMHC alleles. This is useful in a variety of contexts. One specific usecase for the presentation identification system 160 is that it is ableto receive nucleotide sequences of candidate neoantigens associated witha set of MHC alleles from tumor cells of a patient 110 and determinelikelihoods that the candidate neoantigens will be presented by one ormore of the associated MHC alleles of the tumor and/or induceimmunogenic responses in the immune system of the patient 110. Thosecandidate neoantigens with high likelihoods as determined by system 160can be selected for inclusion in a vaccine 118, such an anti-tumorimmune response can be elicited from the immune system of the patient110 providing the tumor cells. Additionally, T-cells with TCRs that areresponsive to candidate neoantigens with high presentation likelihoodscan be produced for use in T-cell therapy, thereby also eliciting ananti-tumor immune response from the immune system of the patient 110.

The presentation identification system 160 determines presentationlikelihoods through one or more presentation models. Specifically, thepresentation models generate likelihoods of whether given peptidesequences will be presented for a set of associated MHC alleles, and aregenerated based on presentation information stored in store 165. Forexample, the presentation models may generate likelihoods of whether apeptide sequence “YVYVADVAAK (SEQ ID NO: 1)” will be presented for theset of alleles HLA-A*02:01, HLA-A*03:01, HLA-B*07:02, HLA-B*08:03,HLA-C*01:04 on the cell surface of the sample. The presentationinformation 165 contains information on whether peptides bind todifferent types of MHC alleles such that those peptides are presented byMHC alleles, which in the models is determined depending on positions ofamino acids in the peptide sequences. The presentation model can predictwhether an unrecognized peptide sequence will be presented inassociation with an associated set of MHC alleles based on thepresentation information 165. As previously mentioned, the presentationmodels may be applied to both class I and class II MHC alleles.

VII.B. Presentation Information

FIG. 2 illustrates a method of obtaining presentation information, inaccordance with an embodiment. The presentation information 165 includestwo general categories of information: allele-interacting informationand allele-noninteracting information. Allele-interacting informationincludes information that influence presentation of peptide sequencesthat are dependent on the type of MHC allele. Allele-noninteractinginformation includes information that influence presentation of peptidesequences that are independent on the type of MHC allele.

VII.B.1. Allele-Interacting Information

Allele-interacting information primarily includes identified peptidesequences that are known to have been presented by one or moreidentified MHC molecules from humans, mice, etc. Notably, this may ormay not include data obtained from tumor samples. The presented peptidesequences may be identified from cells that express a single MHC allele.In this case the presented peptide sequences are generally collectedfrom single-allele cell lines that are engineered to express apredetermined MHC allele and that are subsequently exposed to syntheticprotein. Peptides presented on the MHC allele are isolated by techniquessuch as acid-elution and identified through mass spectrometry. FIG. 2Bshows an example of this, where the example peptide YEMFNDKSQRAPDDKMF(SEQ ID NO: 2), presented on the predetermined MHC alleleHLA-DRB1*12:01, is isolated and identified through mass spectrometry.Since in this situation peptides are identified through cells engineeredto express a single predetermined MHC protein, the direct associationbetween a presented peptide and the MHC protein to which it was bound tois definitively known.

The presented peptide sequences may also be collected from cells thatexpress multiple MHC alleles. Typically in humans, 6 different types ofMHC-I and up to 12 different types of MHC-II molecules are expressed fora cell. Such presented peptide sequences may be identified frommultiple-allele cell lines that are engineered to express multiplepredetermined MHC alleles. Such presented peptide sequences may also beidentified from tissue samples, either from normal tissue samples ortumor tissue samples. In this case particularly, the MHC molecules canbe immunoprecipitated from normal or tumor tissue. Peptides presented onthe multiple MHC alleles can similarly be isolated by techniques such asacid-elution and identified through mass spectrometry FIG. 2C shows anexample of this, where the six example peptides, YEMFNDKSF (SEQ ID NO:3), HROEIFSHDFJ (SEQ ID NO: 4), FJIEJFOESS (SEQ ID NO: 5), NEIOREIREI(SEQ ID NO: 6), JFKSIFEMMSJDSSUIFLKSJFIEIFJ (SEQ ID NO: 7), andKNFLENFIESOFI (SEQ ID NO: 8), are presented on identified class I MHCalleles HLA-A*01:01, HLA-A*02:01, HLA-B*07:02, HLA-B*08:01, and class IIMHC alleles HLA-DRB1*10:01, HLA-DRB1:11:0 land are isolated andidentified through mass spectrometry. In contrast to single-allele celllines, the direct association between a presented peptide and the MHCprotein to which it was bound to may be unknown since the bound peptidesare isolated from the MHC molecules before being identified.

Allele-interacting information can also include mass spectrometry ioncurrent which depends on both the concentration of peptide-MHC moleculecomplexes, and the ionization efficiency of peptides. The ionizationefficiency varies from peptide to peptide in a sequence-dependentmanner. Generally, ionization efficiency varies from peptide to peptideover approximately two orders of magnitude, while the concentration ofpeptide-MHC complexes varies over a larger range than that.

Allele-interacting information can also include measurements orpredictions of binding affinity between a given MHC allele and a givenpeptide. (72, 73, 74) One or more affinity models can generate suchpredictions. For example, going back to the example shown in FIG. 1D,presentation information 165 may include a binding affinity predictionof 1000 nM between the peptide YEMFNDKSF (SEQ ID NO: 3) and the class Iallele HLA-A*01:01. Few peptides with IC50>1000 nm are presented by theMHC, and lower IC50 values increase the probability of presentation.Presentation information 165 may include a binding affinity predictionbetween the peptide KNFLENFIESOFI (SEQ ID NO: 8) and the class II alleleHLA-DRB1:11:01.

Allele-interacting information can also include measurements orpredictions of stability of the MHC complex. One or more stabilitymodels that can generate such predictions. More stable peptide-MHCcomplexes (i.e., complexes with longer half-lives) are more likely to bepresented at high copy number on tumor cells and on antigen-presentingcells that encounter vaccine antigen. For example, going back to theexample shown in FIG. 2C, presentation information 165 may include astability prediction of a half-life of 1 h for the class I moleculeHLA-A*01:01. Presentation information 165 may also include a stabilityprediction of a half-life for the class II molecule HLA-DRB1:11:01.

Allele-interacting information can also include the measured orpredicted rate of the formation reaction for the peptide-MHC complex.Complexes that form at a higher rate are more likely to be presented onthe cell surface at high concentration.

Allele-interacting information can also include the sequence and lengthof the peptide. MHC class I molecules typically prefer to presentpeptides with lengths between 8 and 15 peptides. 60-80% of presentedpeptides have length 9. MHC class II molecules typically prefer topresent peptides with lengths between 6-30 peptides.

Allele-interacting information can also include the presence of kinasesequence motifs on the neoantigen encoded peptide, and the absence orpresence of specific post-translational modifications on the neoantigenencoded peptide. The presence of kinase motifs affects the probabilityof post-translational modification, which may enhance or interfere withMHC binding.

Allele-interacting information can also include the expression oractivity levels of proteins involved in the process ofpost-translational modification, e.g., kinases (as measured or predictedfrom RNA seq, mass spectrometry, or other methods).

Allele-interacting information can also include the probability ofpresentation of peptides with similar sequence in cells from otherindividuals expressing the particular MHC allele as assessed bymass-spectrometry proteomics or other means.

Allele-interacting information can also include the expression levels ofthe particular MHC allele in the individual in question (e.g. asmeasured by RNA-seq or mass spectrometry). Peptides that bind moststrongly to an MHC allele that is expressed at high levels are morelikely to be presented than peptides that bind most strongly to an MHCallele that is expressed at a low level.

Allele-interacting information can also include the overall neoantigenencoded peptide-sequence-independent probability of presentation by theparticular MHC allele in other individuals who express the particularMHC allele.

Allele-interacting information can also include the overallpeptide-sequence-independent probability of presentation by MHC allelesin the same family of molecules (e.g., HLA-A, HLA-B, HLA-C, HLA-DQ,HLA-DR, HLA-DP) in other individuals. For example, HLA-C molecules aretypically expressed at lower levels than HLA-A or HLA-B molecules, andconsequently, presentation of a peptide by HLA-C is a priori lessprobable than presentation by HLA-A or HLA-B. For another example,HLA-DP is typically expressed at lower levels than HLA-DR or HLA-DQ;consequently, presentation of a peptide by HLA-DP is a prior lessprobable than presentation by HLA-DR or HLA-DQ.

Allele-interacting information can also include the protein sequence ofthe particular MHC allele.

Any MHC allele-noninteracting information listed in the below sectioncan also be modeled as an MHC allele-interacting information.

VII.B.2. Allele-noninteracting Information

Allele-noninteracting information can include C-terminal sequencesflanking the neoantigen encoded peptide within its source proteinsequence. For MHC-I, C-terminal flanking sequences may impactproteasomal processing of peptides. However, the C-terminal flankingsequence is cleaved from the peptide by the proteasome before thepeptide is transported to the endoplasmic reticulum and encounters MHCalleles on the surfaces of cells. Consequently, MHC molecules receive noinformation about the C-terminal flanking sequence, and thus, the effectof the C-terminal flanking sequence cannot vary depending on MHC alleletype. For example, going back to the example shown in FIG. 2C,presentation information 165 may include the C-terminal flankingsequence FOEIFNDKSLDKFJI (SEQ ID NO: 9) of the presented peptideFJIEJFOESS (SEQ ID NO: 5) identified from the source protein of thepeptide.

Allele-noninteracting information can also include mRNA quantificationmeasurements. For example, mRNA quantification data can be obtained forthe same samples that provide the mass spectrometry training data. Aslater described in reference to FIG. 13H, RNA expression was identifiedto be a strong predictor of peptide presentation. In one embodiment, themRNA quantification measurements are identified from software tool RSEM.Detailed implementation of the RSEM software tool can be found at Bo Liand Colin N. Dewey. RSEM: accurate transcript quantification fromRNA-Seq data with or without a reference genome. BMC Bioinformatics,12:323, August 2011. In one embodiment, the mRNA quantification ismeasured in units of fragments per kilobase of transcript per Millionmapped reads (FPKM).

Allele-noninteracting information can also include the N-terminalsequences flanking the peptide within its source protein sequence.

Allele-noninteracting information can also include the source gene ofthe peptide sequence. The source gene may be defined as the Ensemblprotein family of the peptide sequence. In other examples, the sourcegene may be defined as the source DNA or the source RNA of the peptidesequence. The source gene can, for example, be represented as a stringof nucleotides that encode for a protein, or alternatively be morecategorically represented based on a named set of known DNA or RNAsequences that are known to encode specific proteins. In anotherexample, allele-noninteracting information can also include the sourcetranscript or isoform or set of potential source transcripts or isoformsof the peptide sequence drawn from a database such as Ensembl or RefSeq.

Allele-noninteracting information can also include the tissue type, celltype or tumor type of cells of origin of the peptide sequence.

Allele-noninteracting information can also include the presence ofprotease cleavage motifs in the peptide, optionally weighted accordingto the expression of corresponding proteases in the tumor cells (asmeasured by RNA-seq or mass spectrometry). Peptides that containprotease cleavage motifs are less likely to be presented, because theywill be more readily degraded by proteases, and will therefore be lessstable within the cell.

Allele-noninteracting information can also include the turnover rate ofthe source protein as measured in the appropriate cell type. Fasterturnover rate (i.e., lower half-life) increases the probability ofpresentation; however, the predictive power of this feature is low ifmeasured in a dissimilar cell type.

Allele-noninteracting information can also include the length of thesource protein, optionally considering the specific splice variants(“isoforms”) most highly expressed in the tumor cells as measured byRNA-seq or proteome mass spectrometry, or as predicted from theannotation of germline or somatic splicing mutations detected in DNA orRNA sequence data.

Allele-noninteracting information can also include the level ofexpression of the proteasome, immunoproteasome, thymoproteasome, orother proteases in the tumor cells (which may be measured by RNA-seq,proteome mass spectrometry, or immunohistochemistry). Differentproteasomes have different cleavage site preferences. More weight willbe given to the cleavage preferences of each type of proteasome inproportion to its expression level.

Allele-noninteracting information can also include the expression of thesource gene of the peptide (e.g., as measured by RNA-seq or massspectrometry). Possible optimizations include adjusting the measuredexpression to account for the presence of stromal cells andtumor-infiltrating lymphocytes within the tumor sample. Peptides frommore highly expressed genes are more likely to be presented. Peptidesfrom genes with undetectable levels of expression can be excluded fromconsideration.

Allele-noninteracting information can also include the probability thatthe source mRNA of the neoantigen encoded peptide will be subject tononsense-mediated decay as predicted by a model of nonsense-mediateddecay, for example, the model from Rivas et al. Science 2015.

Allele-noninteracting information can also include the typicaltissue-specific expression of the source gene of the peptide duringvarious stages of the cell cycle. Genes that are expressed at a lowlevel overall (as measured by RNA-seq or mass spectrometry proteomics)but that are known to be expressed at a high level during specificstages of the cell cycle are likely to produce more presented peptidesthan genes that are stably expressed at very low levels.

Allele-noninteracting information can also include a comprehensivecatalog of features of the source protein as given in e.g. uniProt orPDB http://www.rcsb.org/pdbhome/home.do. These features may include,among others: the secondary and tertiary structures of the protein,subcellular localization 11. Gene ontology (GO) terms. Specifically,this information may contain annotations that act at the level of theprotein, e.g., 5′ UTR length, and annotations that act at the level ofspecific residues, e.g., helix motif between residues 300 and 310. Thesefeatures can also include turn motifs, sheet motifs, and disorderedresidues.

Allele-noninteracting information can also include features describingthe properties of the domain of the source protein containing thepeptide, for example: secondary or tertiary structure (e.g., alpha helixvs beta sheet); Alternative splicing.

Allele-noninteracting information can also include features describingthe presence or absence of a presentation hotspot at the position of thepeptide in the source protein of the peptide.

Allele-noninteracting information can also include the probability ofpresentation of peptides from the source protein of the peptide inquestion in other individuals (after adjusting for the expression levelof the source protein in those individuals and the influence of thedifferent HLA types of those individuals).

Allele-noninteracting information can also include the probability thatthe peptide will not be detected or over-represented by massspectrometry due to technical biases.

The expression of various gene modules/pathways as measured by a geneexpression assay such as RNASeq, microarray(s), targeted panel(s) suchas Nanostring, or single/multi-gene representatives of gene modulesmeasured by assays such as RT-PCR (which need not contain the sourceprotein of the peptide) that are informative about the state of thetumor cells, stroma, or tumor-infiltrating lymphocytes (TILs).

Allele-noninteracting information can also include the copy number ofthe source gene of the peptide in the tumor cells. For example, peptidesfrom genes that are subject to homozygous deletion in tumor cells can beassigned a probability of presentation of zero.

Allele-noninteracting information can also include the probability thatthe peptide binds to the TAP or the measured or predicted bindingaffinity of the peptide to the TAP. Peptides that are more likely tobind to the TAP, or peptides that bind the TAP with higher affinity aremore likely to be presented by MHC-I.

Allele-noninteracting information can also include the expression levelof TAP in the tumor cells (which may be measured by RNA-seq, proteomemass spectrometry, immunohistochemistry). For MHC-I, higher TAPexpression levels increase the probability of presentation of allpeptides.

Allele-noninteracting information can also include the presence orabsence of tumor mutations, including, but not limited to:

-   -   i. Driver mutations in known cancer driver genes such as EGFR,        KRAS, ALK, RET, ROS1, TP53, CDKN2A, CDKN2B, NTRK1, NTRK2, NTRK3    -   ii. In genes encoding the proteins involved in the antigen        presentation machinery (e.g., B2M, HLA-A, HLA-B, HLA-C, TAP-1,        TAP-2, TAPBP, CALR, CNX, ERP57, HLA-DM, HLA-DMA, HLA-DMB,        HLA-DO, HLA-DOA, HLA-DOBHLA-DP, HLA-DPA1, HLA-DPB1, HLA-DQ,        HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DR, HLA-DRA,        HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5 or any of the genes        coding for components of the proteasome or immunoproteasome).        Peptides whose presentation relies on a component of the        antigen-presentation machinery that is subject to        loss-of-function mutation in the tumor have reduced probability        of presentation.

Presence or absence of functional germline polymorphisms, including, butnot limited to:

-   -   i. In genes encoding the proteins involved in the antigen        presentation machinery (e.g., B2M, HLA-A, HLA-B, HLA-C, TAP-1,        TAP-2, TAPBP, CALR, CNX, ERP57, HLA-DM, HLA-DMA, HLA-DMB,        HLA-DO, HLA-DOA, HLA-DOBHLA-DP, HLA-DPA1, HLA-DPB1, HLA-DQ,        HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DR, HLA-DRA,        HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5 or any of the genes        coding for components of the proteasome or immunoproteasome)

Allele-noninteracting information can also include tumor type (e.g.,NSCLC, melanoma).

Allele-noninteracting information can also include known functionalityof HLA alleles, as reflected by, for instance HLA allele suffixes. Forexample, the N suffix in the allele name HLA-A*24:09N indicates a nullallele that is not expressed and is therefore unlikely to presentepitopes; the full HLA allele suffix nomenclature is described athttps://www.ebi.ac.uk/ipd/imgt/hla/nomenclature/suffixes.html.

Allele-noninteracting information can also include clinical tumorsubtype (e.g., squamous lung cancer vs. non-squamous).

Allele-noninteracting information can also include smoking history.

Allele-noninteracting information can also include history of sunburn,sun exposure, or exposure to other mutagens.

Allele-noninteracting information can also include the typicalexpression of the source gene of the peptide in the relevant tumor typeor clinical subtype, optionally stratified by driver mutation. Genesthat are typically expressed at high levels in the relevant tumor typeare more likely to be presented.

Allele-noninteracting information can also include the frequency of themutation in all tumors, or in tumors of the same type, or in tumors fromindividuals with at least one shared MHC allele, or in tumors of thesame type in individuals with at least one shared MHC allele.

In the case of a mutated tumor-specific peptide, the list of featuresused to predict a probability of presentation may also include theannotation of the mutation (e.g., missense, read-through, frameshift,fusion, etc.) or whether the mutation is predicted to result innonsense-mediated decay (NMD). For example, peptides from proteinsegments that are not translated in tumor cells due to homozygousearly-stop mutations can be assigned a probability of presentation ofzero. NMD results in decreased mRNA translation, which decreases theprobability of presentation.

VII.C. Presentation Identification System

FIG. 3 is a high-level block diagram illustrating the computer logiccomponents of the presentation identification system 160, according toone embodiment. In this example embodiment, the presentationidentification system 160 includes a data management module 312, anencoding module 314, a training module 316, and a prediction module 320.The presentation identification system 160 is also comprised of atraining data store 170 and a presentation models store 175. Someembodiments of the model management system 160 have different modulesthan those described here. Similarly, the functions can be distributedamong the modules in a different manner than is described here.

VII.C.1. Data Management Module

The data management module 312 generates sets of training data 170 fromthe presentation information 165. Each set of training data contains aplurality of data instances, in which each data instance i contains aset of independent variables z^(i) that include at least a presented ornon-presented peptide sequence p^(i), one or more associated MHC allelesa^(i) associated with the peptide sequence p^(i), and a dependentvariable y^(i) that represents information that the presentationidentification system 160 is interested in predicting for new values ofindependent variables.

In one particular implementation referred throughout the remainder ofthe specification, the dependent variable y^(i) is a binary labelindicating whether peptide p^(i) was presented by the one or moreassociated MHC alleles a^(i). However, it is appreciated that in otherimplementations, the dependent variable) can represent any other kind ofinformation that the presentation identification system 160 isinterested in predicting dependent on the independent variables z^(i).For example, in another implementation, the dependent variable y^(i) mayalso be a numerical value indicating the mass spectrometry ion currentidentified for the data instance.

The peptide sequence p^(i) for data instance i is a sequence of k_(i)amino acids, in which k_(i) may vary between data instances i within arange. For example, that range may be 8-15 for MHC class I or 6-30 forMHC class II. In one specific implementation of system 160, all peptidesequences p^(i) in a training data set may have the same length, e.g. 9.The number of amino acids in a peptide sequence may vary depending onthe type of MHC alleles (e.g., MHC alleles in humans, etc.). The MHCalleles a^(i) for data instance i indicate which MHC alleles werepresent in association with the corresponding peptide sequence p^(i).

The data management module 312 may also include additionalallele-interacting variables, such as binding affinity b^(i) andstability s^(i) predictions in conjunction with the peptide sequencesp^(i) and associated MHC alleles a^(i) contained in the training data170. For example, the training data 170 may contain binding affinitypredictions b^(i) between a peptide p^(i) and each of the associated MHCmolecules indicated in a^(i). As another example, the training data 170may contain stability predictions s^(i) for each of the MHC allelesindicated in a^(i).

The data management module 312 may also include allele-noninteractingvariables w^(i), such as C-terminal flanking sequences and mRNAquantification measurements in conjunction with the peptide sequencesp^(i).

The data management module 312 also identifies peptide sequences thatare not presented by MHC alleles to generate the training data 170.Generally, this involves identifying the “longer” sequences of sourceprotein that include presented peptide sequences prior to presentation.When the presentation information contains engineered cell lines, thedata management module 312 identifies a series of peptide sequences inthe synthetic protein to which the cells were exposed to that were notpresented on MHC alleles of the cells. When the presentation informationcontains tissue samples, the data management module 312 identifiessource proteins from which presented peptide sequences originated from,and identifies a series of peptide sequences in the source protein thatwere not presented on MHC alleles of the tissue sample cells.

The data management module 312 may also artificially generate peptideswith random sequences of amino acids and identify the generatedsequences as peptides not presented on MHC alleles. This can beaccomplished by randomly generating peptide sequences allows the datamanagement module 312 to easily generate large amounts of synthetic datafor peptides not presented on MHC alleles. Since in reality, a smallpercentage of peptide sequences are presented by MHC alleles, thesynthetically generated peptide sequences are highly likely not to havebeen presented by MHC alleles even if they were included in proteinsprocessed by cells.

FIG. 4 illustrates an example set of training data 170A, according toone embodiment. Specifically, the first 3 data instances in the trainingdata 170A indicate peptide presentation information from a single-allelecell line involving the allele HLA-C*01:03 and 3 peptide sequencesQCEIOWAREFLKEIGJ (SEQ ID NO: 10), FIEUHFWI (SEQ ID NO: 11), andFEWRHRJTRUJR (SEQ ID NO: 12). The fourth data instance in the trainingdata 170A indicates peptide information from a multiple-allele cell lineinvolving the alleles HLA-B*07:02, HLA-C*01:03, HLA-A*01:01 and apeptide sequence QIEJOEIJE (SEQ ID NO: 13). The first data instanceindicates that peptide sequence QCEIOWARE (SEQ ID NO: 14) was notpresented by the allele HLA-DRB3:01:01. As discussed in the prior twoparagraphs, the negatively-labeled peptide sequences may be randomlygenerated by the data management module 312 or identified from sourceprotein of presented peptides. The training data 170A also includes abinding affinity prediction of 1000 nM and a stability prediction of ahalf-life of 1 h for the peptide sequence-allele pair. The training data170A also includes allele-noninteracting variables, such as theC-terminal flanking sequence of the peptide FJELFISBOSJFIE (SEQ ID NO:15), and a mRNA quantification measurement of 10² TPM. The fourth datainstance indicates that peptide sequence QIEJOEIJE (SEQ ID NO: 13) waspresented by one of the alleles HLA-B*07:02, HLA-C*01:03, orHLA-A*01:01. The training data 170A also includes binding affinitypredictions and stability predictions for each of the alleles, as wellas the C-terminal flanking sequence of the peptide and the mRNAquantification measurement for the peptide.

VII.C.2. Encoding Module

The encoding module 314 encodes information contained in the trainingdata 170 into a numerical representation that can be used to generatethe one or more presentation models. In one implementation, the encodingmodule 314 one-hot encodes sequences (e.g., peptide sequences orC-terminal flanking sequences) over a predetermined 20-letter amino acidalphabet. Specifically, a peptide sequence p^(i) with k_(i) amino acidsis represented as a row vector of 20·k_(i) elements, where a singleelement among p^(i) _(20·(j−1)+1), p^(i) _(20·(j−1)+2), . . . , p^(i)_(20·j), that corresponds to the alphabet of the amino acid at the j-thposition of the peptide sequence has a value of 1. Otherwise, theremaining elements have a value of 0. As an example, for a givenalphabet {A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y},the peptide sequence EAF of 3 amino acids for data instance i may berepresented by the row vector of 60 elements p^(i)=10 0 0 1 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 00 0 0 0 0 0 0 0 0 0 0 0 0 0 1. The C-terminal flanking sequence c can besimilarly encoded as described above, as well as the protein sequenced_(h) for MHC alleles, and other sequence data in the presentationinformation.

When the training data 170 contains sequences of differing lengths ofamino acids, the encoding module 314 may further encode the peptidesinto equal-length vectors by adding a PAD character to extend thepredetermined alphabet. For example, this may be performed byleft-padding the peptide sequences with the PAD character until thelength of the peptide sequence reaches the peptide sequence with thegreatest length in the training data 170. Thus, when the peptidesequence with the greatest length has k_(max) amino acids, the encodingmodule 314 numerically represents each sequence as a row vector of(20+)·k_(max) elements. As an example, for the extended alphabet {PAD,A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y} and amaximum amino acid length of k_(max)=5, the same example peptidesequence EAF of 3 amino acids may be represented by the row vector of105 elements p^(i)=[1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0].The C-terminal flanking sequence c or other sequence data can besimilarly encoded as described above. Thus, each independent variable orcolumn in the peptide sequence p^(i) or c^(i) represents presence of aparticular amino acid at a particular position of the sequence.

Although the above method of encoding sequence data was described inreference to sequences having amino acid sequences, the method cansimilarly be extended to other types of sequence data, such as DNA orRNA sequence data, and the like.

The encoding module 314 also encodes the one or more MHC alleles a^(i)for data instance i as a row vector of m elements, in which each elementh=1, 2, . . . , m corresponds to a unique identified MHC allele. Theelements corresponding to the MHC alleles identified for the datainstance i have a value of 1. Otherwise, the remaining elements have avalue of 0. As an example, the alleles HLA-1B*07:02 and HLA-DRB*10:01for a data instance i corresponding to a multiple-allele cell line amongm=4 unique identified MHC allele types {HLA-A*01:01, HLA-C*01:08,HLA-1B*07:02, HLA-DRB1*10:01} may be represented by the row vector of 4elements a^(i)=[0 0 1 1], in which a₃ ^(i)=1 and a₄ ^(i)=1. Although theexample is described herein with 4 identified MHC allele types, thenumber of MHC allele types can be hundreds or thousands in practice. Aspreviously discussed, each data instance i typically contains at most 6different MHC allele types in association with the peptide sequencep^(i).

The encoding module 314 also encodes the label y_(i) for each datainstance i as a binary variable having values from the set of {0, 1}, inwhich a value of 1 indicates that peptide x^(i) was presented by one ofthe associated MHC alleles a^(i), and a value of 0 indicates thatpeptide x^(i) was not presented by any of the associated MHC allelesa^(i). When the dependent variable y_(i) represents the massspectrometry ion current, the encoding module 314 may additionally scalethe values using various functions, such as the log function having arange of (−∞, ∞) for ion current values between [0, ∞).

The encoding module 314 may represent a pair of allele-interactingvariables x_(h) ^(i) for peptide p_(i) and an associated MHC allele h asa row vector in which numerical representations of allele-interactingvariables are concatenated one after the other. For example, theencoding module 314 may represent x_(h) ^(i) as a row vector equal to[p^(i)], [p^(i) b_(h) ^(i)], [p^(i) s_(h) ^(i)], or [p^(i) b_(h) ^(i)s_(h) ^(i)], where b_(h) ^(i) is the binding affinity prediction forpeptide p_(i) and associated MHC allele h, and similarly for s_(h) ^(i)for stability. Alternatively, one or more combination ofallele-interacting variables may be stored individually (e.g., asindividual vectors or matrices).

In one instance, the encoding module 314 represents binding affinityinformation by incorporating measured or predicted values for bindingaffinity in the allele-interacting variables x_(h) ^(i).

In one instance, the encoding module 314 represents binding stabilityinformation by incorporating measured or predicted values for bindingstability in the allele-interacting variables x_(h) ^(i),

In one instance, the encoding module 314 represents binding on-rateinformation by incorporating measured or predicted values for bindingon-rate in the allele-interacting variables x_(h) ^(i).

In one instance, for peptides presented by class I MHC molecules, theencoding module 314 represents peptide length as a vector T_(k)=[

(L_(k)=8)

(L_(k)=9)

(L_(k)=10)

(L_(k)=11)

(L_(k)=12)

(L_(k)=13)

(L_(k)=14)

(L=15)] where

is the indicator function, and L_(k) denotes the length of peptidep_(k). The vector T_(k) can be included in the allele-interactingvariables x_(h) ^(i). In another instance, for peptides presented byclass II MHC molecules, the encoding module 314 represents peptidelength as a vector T_(k)=[

(L_(k)=6)

(L_(k)=7)

(L_(k)=8)

(L_(k)=9)

(L_(k)=10)

(L_(k)=11)

(L_(k)=12)

(L_(k)=13)

(L_(k)=14)

(L=15)

(L_(k)=16)

(L=17)

(L=18)

(L_(k)=19)

(L_(k)=20)

(L_(k)=21)

(L_(k)=22)

(L_(k)=23)

(L_(k)=24)

(L_(k)=25)

(L_(k)=26)

(L_(k)=27)

(L_(k)=28)

(L_(k)=29)

(L_(k)=30)] where

is the indicator function, and L_(k) denotes the length of peptidep_(k). The vector T_(k) can be included in the allele-interactingvariables x_(h) ^(i).

In one instance, the encoding module 314 represents RNA expressioninformation of MHC alleles by incorporating RNA-seq based expressionlevels of MHC alleles in the allele-interacting variables x_(h) ^(i).

Similarly, the encoding module 314 may represent theallele-noninteracting variables w^(i) as a row vector in which numericalrepresentations of allele-noninteracting variables are concatenated oneafter the other. For example, w^(i) may be a row vector equal to [c^(i)]or [c^(i) m^(i) w^(i)] in which w is a row vector representing any otherallele-noninteracting variables in addition to the C-terminal flankingsequence of peptide p^(i) and the mRNA quantification measurement m^(i)associated with the peptide. Alternatively, one or more combination ofallele-noninteracting variables may be stored individually (e.g., asindividual vectors or matrices).

In one instance, the encoding module 314 represents turnover rate ofsource protein for a peptide sequence by incorporating the turnover rateor half-life in the allele-noninteracting variables w^(i).

In one instance, the encoding module 314 represents length of sourceprotein or isoform by incorporating the protein length in theallele-noninteracting variables w^(i).

In one instance, the encoding module 314 represents activation ofimmunoproteasome by incorporating the mean expression of theimmunoproteasome-specific proteasome subunits including the β1 _(i), β2_(i), β5 _(i) subunits in the allele-noninteracting variables w^(i).

In one instance, the encoding module 314 represents the RNA-seqabundance of the source protein of the peptide or gene or transcript ofa peptide (quantified in units of FPKM, TPM by techniques such as RSEM)can be incorporating the abundance of the source protein in theallele-noninteracting variables w^(i).

In one instance, the encoding module 314 represents the probability thatthe transcript of origin of a peptide will undergo nonsense-mediateddecay (NMD) as estimated by the model in, for example, Rivas et. al.Science, 2015 by incorporating this probability in theallele-noninteracting variables w^(i).

In one instance, the encoding module 314 represents the activationstatus of a gene module or pathway assessed via RNA-seq by, for example,quantifying expression of the genes in the pathway in units of TPM usinge.g., RSEM for each of the genes in the pathway then computing a summarystatistics, e.g., the mean, across genes in the pathway. The mean can beincorporated in the allele-noninteracting variables w.

In one instance, the encoding module 314 represents the copy number ofthe source gene by incorporating the copy number in theallele-noninteracting variables w^(i).

In one instance, the encoding module 314 represents the TAP bindingaffinity by including the measured or predicted TAP binding affinity(e.g., in nanomolar units) in the allele-noninteracting variables w^(i).

In one instance, the encoding module 314 represents TAP expressionlevels by including TAP expression levels measured by RNA-seq (andquantified in units of TPM by e.g., RSEM) in the allele-noninteractingvariables w^(i).

In one instance, the encoding module 314 represents tumor mutations as avector of indicator variables (i.e., d^(k)=1 if peptide p^(k) comes froma sample with a KRAS G12D mutation and 0 otherwise) in theallele-noninteracting variables w^(i).

In one instance, the encoding module 314 represents germlinepolymorphisms in antigen presentation genes as a vector of indicatorvariables (i.e., d=1 if peptide p^(k) comes from a sample with aspecific germline polymorphism in the TAP). These indicator variablescan be included in the allele-noninteracting variables w^(i).

In one instance, the encoding module 314 represents tumor type as alength-one one-hot encoded vector over the alphabet of tumor types(e.g., NSCLC, melanoma, colorectal cancer, etc). These one-hot-encodedvariables can be included in the allele-noninteracting variables w^(i).

In one instance, the encoding module 314 represents MHC allele suffixesby treating 4-digit HLA alleles with different suffixes. For example,HLA-A*24:09N is considered a different allele from HLA-A*24:09 for thepurpose of the model. Alternatively, the probability of presentation byan N-suffixed MHC allele can be set to zero for all peptides, becauseHLA alleles ending in the N suffix are not expressed.

In one instance, the encoding module 314 represents tumor subtype as alength-one one-hot encoded vector over the alphabet of tumor subtypes(e.g., lung adenocarcinoma, lung squamous cell carcinoma, etc). Theseone-hot encoded variables can be included in the allele-noninteractingvariables w.

In one instance, the encoding module 314 represents smoking history as abinary indicator variable (d^(k)=1 if the patient has a smoking history,and 0 otherwise), that can be included in the allele-noninteractingvariables w^(i). Alternatively, smoking history can be encoded as alength-one one-hot encoded variable over an alphabet of smokingseverity. For example, smoking status can be rated on a 1-5 scale, where1 indicates nonsmokers, and 5 indicates current heavy smokers. Becausesmoking history is primarily relevant to lung tumors, when training amodel on multiple tumor types, this variable can also be defined to beequal to 1 if the patient has a history of smoking and the tumor type islung tumors and zero otherwise.

In one instance, the encoding module 314 represents sunburn history as abinary indicator variable (d^(k)=1 if the patient has a history ofsevere sunburn, and 0 otherwise), which can be included in theallele-noninteracting variables w^(i). Because severe sunburn isprimarily relevant to melanomas, when training a model on multiple tumortypes, this variable can also be defined to be equal to 1 if the patienthas a history of severe sunburn and the tumor type is melanoma and zerootherwise.

In one instance, the encoding module 314 represents distribution ofexpression levels of a particular gene or transcript for each gene ortranscript in the human genome as summary statistics (e.g., mean,median) of distribution of expression levels by using referencedatabases such as TCGA. Specifically, for a peptide p^(k) in a samplewith tumor type melanoma, not only the measured gene or transcriptexpression level of the gene or transcript of origin of peptide p^(k) inthe allele-noninteracting variables w^(i), but also the mean and/ormedian gene or transcript expression of the gene or transcript of originof peptide p^(k) in melanomas as measured by TCGA can be included.

In one instance, the encoding module 314 represents mutation type as alength-one one-hot-encoded variable over the alphabet of mutation types(e.g., missense, frameshift, NMD-inducing, etc). These onehot-encodedvariables can be included in the allele-noninteracting variables w^(i).

In one instance, the encoding module 314 represents protein-levelfeatures of protein as the value of the annotation (e.g., 5′ UTR length)of the source protein in the allele-noninteracting variables w^(i). Inanother instance, the encoding module 314 represents residue-levelannotations of the source protein for peptide p^(i) by including anindicator variable, that is equal to 1 if peptide p^(i) overlaps with ahelix motif and 0 otherwise, or that is equal to 1 if peptide p^(i) iscompletely contained with within a helix motif in theallele-noninteracting variables w^(i). In another instance, a featurerepresenting proportion of residues in peptide p^(i) that are containedwithin a helix motif annotation can be included in theallele-noninteracting variables w^(i).

In one instance, the encoding module 314 represents type of proteins orisoforms in the human proteome as an indicator vector o^(k) that has alength equal to the number of proteins or isoforms in the humanproteome, and the corresponding element o^(k) _(i) is 1 if peptide p^(k)comes from protein i and 0 otherwise.

In one instance, the encoding module 314 represents the source geneG=gene(p^(i)) of peptide p^(i) as a categorical variable with L possiblecategories, where L denotes the upper limit of the number of indexedsource genes 1, 2, . . . , L.

In one instance, the encoding module 314 represents the tissue type,cell type, tumor type, or tumor histology type T=tissue(p^(i)) ofpeptide p^(i) as a categorical variable with M possible categories,where M denotes the upper limit of the number of indexed types 1, 2, . .. , M. Types of tissue can include, for example, lung tissue, cardiactissue, intestine tissue, nerve tissue, and the like. Types of cells caninclude dendritic cells, macrophages, CD4 T cells, and the like. Typesof tumors can include lung adenocarcinoma, lung squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, and the like.

The encoding module 314 may also represent the overall set of variablesz^(i) for peptide p^(i) and an associated MHC allele h as a row vectorin which numerical representations of the allele-interacting variablesx^(i) and the allele-noninteracting variables w are concatenated oneafter the other. For example, the encoding module 314 may representz_(h) ^(i) as a row vector equal to [x_(h) ^(i) w^(i)] or [w_(i) x_(h)^(i)].

VIII. Training Module

The training module 316 constructs one or more presentation models thatgenerate likelihoods of whether peptide sequences will be presented byMHC alleles associated with the peptide sequences. Specifically, given apeptide sequence p^(k) and a set of MHC alleles a^(k) associated withthe peptide sequence p^(k), each presentation model generates anestimate u_(k) indicating a likelihood that the peptide sequence p^(k)will be presented by one or more of the associated MHC alleles a^(k).

VIII.A. Overview

The training module 316 constructs the one more presentation modelsbased on the training data sets stored in store 170 generated from thepresentation information stored in 165. Generally, regardless of thespecific type of presentation model, all of the presentation modelscapture the dependence between independent variables and dependentvariables in the training data 170 such that a loss function isminimized. Specifically, the loss function l(γ_(i∈S), u_(i∈S); θ)represents discrepancies between values of dependent variables y_(i∈S)for one or more data instances S in the training data 170 and theestimated likelihoods uses for the data instances S generated by thepresentation model. In one particular implementation referred throughoutthe remainder of the specification, the loss function (γ_(i∈S), i_(i∈S),θ) is the negative log likelihood function given by equation (1a) asfollows:

$\begin{matrix}{{\left( {y_{i \in S},{u_{i \in S};\theta}} \right)} = {\sum\limits_{i \in S}{\left( {{y_{i}\log \; u_{i}} + {\left( {1 - y_{i}} \right){\log \left( {1 - u_{i}} \right)}}} \right).}}} & \left( {1a} \right)\end{matrix}$

However, in practice, another loss function may be used. For example,when predictions are made for the mass spectrometry ion current, theloss function is the mean squared loss given by equation 1b as follows:

$\begin{matrix}{{\left( {y_{i \in S},{u_{i \in S};\theta}} \right)} = {\sum\limits_{i \in S}{\left( {{y - u_{i}}}_{2}^{2} \right).}}} & \left( {1b} \right)\end{matrix}$

The presentation model may be a parametric model in which one or moreparameters θ mathematically specify the dependence between theindependent variables and dependent variables. Typically, variousparameters of parametric-type presentation models that minimize the lossfunction (y_(i∈S), u_(i∈S), θ) are determined through gradient-basednumerical optimization algorithms, such as batch gradient algorithms,stochastic gradient algorithms, and the like. Alternatively, thepresentation model may be a non-parametric model in which the modelstructure is determined from the training data 170 and is not strictlybased on a fixed set of parameters.

VII.B. Per-Allele Models

The training module 316 may construct the presentation models to predictpresentation likelihoods of peptides on a per-allele basis. In thiscase, the training module 316 may train the presentation models based ondata instances S in the training data 170 generated from cellsexpressing single MHC alleles.

In one implementation, the training module 316 models the estimatedpresentation likelihood u_(k) for peptide p^(k) for a specific allele hby:

u _(k) ^(h) =Pr(p ^(k) presented;MHC alleleh)=ƒ(g _(h)(x _(h)^(k);θ_(h))),  (2)

where peptide sequence x_(h) ^(k) denotes the encoded allele-interactingvariables for peptide p^(k) and corresponding MHC allele h, ƒ(⋅) is anyfunction, and is herein throughout is referred to as a transformationfunction for convenience of description. Further, g_(h)(⋅) is anyfunction, is herein throughout referred to as a dependency function forconvenience of description, and generates dependency scores for theallele-interacting variables x_(h) ^(k) based on a set of parametersθ_(h) determined for MHC allele h. The values for the set of parametersθ_(h) for each MHC allele h can be determined by minimizing the lossfunction with respect to θ_(h), where i is each instance in the subset Sof training data 170 generated from cells expressing the single MHCallele h.

The output of the dependency function g_(h)(x_(h) ^(k);θ_(h)) representsa dependency score for the MHC allele h indicating whether the MHCallele h will present the corresponding neoantigen based on at least theallele interacting features x_(h) ^(k), and in particular, based onpositions of amino acids of the peptide sequence of peptide p^(k). Forexample, the dependency score for the MHC allele h may have a high valueif the MHC allele h is likely to present the peptide p^(k), and may havea low value if presentation is not likely. The transformation functionƒ(⋅) transforms the input, and more specifically, transforms thedependency score generated by g_(h)(x_(h) ^(k);θ_(h)) in this case, toan appropriate value to indicate the likelihood that the peptide p^(k)will be presented by an MHC allele.

In one particular implementation referred throughout the remainder ofthe specification, ƒ(⋅) is a function having the range within [0, 1] foran appropriate domain range. In one example, ƒ(⋅) is the expit functiongiven by:

$\begin{matrix}{{{f(z)} = \frac{\exp (z)}{1 + {\exp (z)}}}.} & (4)\end{matrix}$

As another example, ƒ(⋅) can also be the hyperbolic tangent functiongiven by:

ƒ(z)=tanh(z)  (5)

when the values for the domain z is equal to or greater than 0.Alternatively, when predictions are made for the mass spectrometry ioncurrent that have values outside the range [0, 1], ƒ(⋅) can be anyfunction such as the identity function, the exponential function, thelog function, and the like.

Thus, the per-allele likelihood that a peptide sequence p^(k) will bepresented by a MHC allele h can be generated by applying the dependencyfunction g_(h)(⋅) for the MHC allele h to the encoded version of thepeptide sequence p^(k) to generate the corresponding dependency score.The dependency score may be transformed by the transformation functionƒ(⋅) to generate a per-allele likelihood that the peptide sequence p^(k)will be presented by the MHC allele h.

VIII.B.1 Dependency Functions for Allele Interacting Variables

In one particular implementation referred throughout the specification,the dependency function g_(h)(⋅) is an affine function given by:

g _(h)(x _(h) ^(i);θ_(h))=x _(h) ^(i)·θ_(h).  (6)

that linearly combines each allele-interacting variable in x_(h) ^(k)with a corresponding parameter in the set of parameters θ_(k) determinedfor the associated MHC allele h.

In another particular implementation referred throughout thespecification, the dependency function g_(h)(⋅) is a network functiongiven by:

g _(h)(x _(h) ^(i);θ_(h))=NN_(h)(x _(h) ^(i);θ_(h))  (7)

represented by a network model NN_(h)(⋅) having a series of nodesarranged in one or more layers. A node may be connected to other nodesthrough connections each having an associated parameter in the set ofparameters θ_(h). A value at one particular node may be represented as asum of the values of nodes connected to the particular node weighted bythe associated parameter mapped by an activation function associatedwith the particular node. In contrast to the affine function, networkmodels are advantageous because the presentation model can incorporatenon-linearity and process data having different lengths of amino acidsequences. Specifically, through non-linear modeling, network models cancapture interaction between amino acids at different positions in apeptide sequence and how this interaction affects peptide presentation.

In general, network models NN_(h)(⋅) may be structured as feed-forwardnetworks, such as artificial neural networks (ANN), convolutional neuralnetworks (CNN), deep neural networks (DNN), and/or recurrent networks,such as long short-term memory networks (LSTM), bi-directional recurrentnetworks, deep bi-directional recurrent networks, and the like.

In one instance referred throughout the remainder of the specification,each MHC allele in h=1, 2 . . . , m is associated with a separatenetwork model, and NN_(h)(⋅) denotes the output(s) from a network modelassociated with MHC allele h.

FIG. 5 illustrates an example network model NN₃(⋅) in association withan arbitrary MHC allele h=3. As shown in FIG. 5, the network modelNN₃(⋅) for MHC allele h=3 includes three input nodes at layer l=1, fournodes at layer l=2, two nodes at layer l=3, and one output node at layer1-4 The network model NN₃(⋅) is associated with a set of ten parametersθ₃(1), θ₃(2), . . . , θ₃(10). The network model NN₃(⋅) receives inputvalues (individual data instances including encoded polypeptide sequencedata and any other training data used) for three allele-interactingvariables x₃ ^(k)(1), x₃ ^(k)(2), and x₃ ^(k)(3) for MHC allele h=3 andoutputs the value NN₃(x₃ ^(k)). The network function may also includeone or more network models each taking different allele interactingvariables as input.

In another instance, the identified MHC alleles h=1, 2, . . . , m areassociated with a single network model NN_(H)(⋅), and NN_(h)(⋅) denotesone or more outputs of the single network model associated with MHCallele h. In such an instance, the set of parameters Ok may correspondto a set of parameters for the single network model, and thus, the setof parameters θ_(h) may be shared by all MHC alleles.

FIG. 6A illustrates an example network model NN_(H)(⋅) shared by MHCalleles h=1, 2, . . . , m. As shown in FIG. 6A, the network modelNN_(N)(⋅) includes m output nodes each corresponding to an MHC allele.The network model NN₃(⋅) receives the allele-interacting variables x-kfor MHC allele h=3 and outputs m values including the value NN₃(x₃ ^(k))corresponding to the MHC allele h=3.

In yet another instance, the single network model NN_(H)(⋅) may be anetwork model that outputs a dependency score given the alleleinteracting variables x_(h) ^(k) and the encoded protein sequence d_(h)of an MHC allele h. In such an instance, the set of parameters θ_(h) mayagain correspond to a set of parameters for the single network model,and thus, the set of parameters θ_(h) may be shared by all MHC alleles.Thus, in such an instance, NN_(h)(⋅) may denote the output of the singlenetwork model NN_(H)(⋅) given inputs [x_(h) ^(k) d_(h)] to the singlenetwork model. Such a network model is advantageous because peptidepresentation probabilities for MHC alleles that were unknown in thetraining data can be predicted just by identification of their proteinsequence.

FIG. 6B illustrates an example network model NN_(H)(⋅) shared by MHCalleles. As shown in FIG. 6B, the network model NN_(H)(⋅) receives theallele interacting variables and protein sequence of MHC allele h=3 asinput, and outputs a dependency score NN₃(x₃ ^(k)) corresponding to theMHC allele h=3.

In yet another instance, the dependency function g_(h)(⋅) can beexpressed as:

_(gh)(x _(h) ^(k);θ_(h))=g′ _(h)(x _(h) ^(k);θ′_(h))+θ_(h) ⁰

where g′_(h)(x_(h) ^(k);θ′_(h)) is the affine function with a set ofparameters θ′_(h), the network function, or the like, with a biasparameter θ_(h) ⁰ in the set of parameters for allele interactingvariables for the MHC allele that represents a baseline probability ofpresentation for the MHC allele h.

In another implementation, the bias parameter θ_(h) ⁰ may be sharedaccording to the gene family of the MHC allele h. That is, the biasparameter θ_(h) ⁰ for MHC allele h may be equal to θ_(gene(h)) ⁰, wheregene(h) is the gene family of MHC allele h. For example, class I MHCalleles HLA-A*02:01, HLA-A*02:02, and HLA-A*02:03 may be assigned to thegene family of “HLA-A” and the bias parameter θ_(h) ⁰ for each of theseMHC alleles may be shared. As another example, class II MHC allelesHLA-DRB1:10:01, HLA-DRB1:11:01, and HLA-DRB3:01:01 may be assigned tothe gene family of “HLA-DRB,” and the bias parameter θ_(h) ⁰ for each ofthese MHC alleles may be shared.

Returning to equation (2), as an example, the likelihood that peptide pwill be presented by MHC allele h=3, among m=4 different identified MHCalleles using the affine dependency function g_(h)(⋅), can be generatedby:

u _(k)=ƒ(x ₃ ^(k)·θ₃),

where x₃ ^(k) are the identified allele-interacting variables for MHCallele h=3, and θ₃ are the set of parameters determined for MHC alleleh=3 through loss function minimization.

As another example, the likelihood that peptide p^(k) will be presentedby MHC allele h=3, among m=4 different identified MHC alleles usingseparate network transformation functions g_(h)(⋅), can be generated by:

u _(k) ³=ƒ(NN₃(x ₃ ^(k);θ₃)),

where x₃ ^(k) are the identified allele-interacting variables for MHCallele h=3, and θ₃ are the set of parameters determined for the networkmodel NN₃(⋅) associated with MHC allele h=3.

FIG. 7 illustrates generating a presentation likelihood for peptidep^(k) in association with MHC allele h=3 using an example network modelNN₃(⋅). As shown in FIG. 7, the network model NN₃(⋅) receives theallele-interacting variables x₃ ^(k) for MHC allele h=3 and generatesthe output NN₃(x₃ ^(k)). The output is mapped by function ƒ(⋅) togenerate the estimated presentation likelihood u_(k).

VIII.B.2. Per-Allele with Allele-Noninteracting Variables

In one implementation, the training module 316 incorporatesallele-noninteracting variables and models the estimated presentationlikelihood u_(k) for peptide P by:

u _(k) ^(h) =Pr(p ^(k) presented)=ƒ(g _(w)(w ^(k);θ_(w))+g ^(h)(x _(h)^(i);θ_(h))),  (8)

where w^(k) denotes the encoded allele-noninteracting variables forpeptide p^(k), g_(w)(⋅) is a function for the allele-noninteractingvariables w^(k) based on a set of parameters θ_(w) determined for theallele-noninteracting variables. Specifically, the values for the set ofparameters θ_(k) for each MHC allele h and the set of parameters θ_(w)for allele-noninteracting variables can be determined by minimizing theloss function with respect to θ_(h) and θ_(w), where i is each instancein the subset S of training data 170 generated from cells expressingsingle MHC alleles.

The output of the dependency function g_(w)(w^(k);θ_(w)) represents adependency score for the allele noninteracting variables indicatingwhether the peptide p^(k) will be presented by one or more MHC allelesbased on the impact of allele noninteracting variables. For example, thedependency score for the allele noninteracting variables may have a highvalue if the peptide p^(k) is associated with a C-terminal flankingsequence that is known to positively impact presentation of the peptidep^(k), and may have a low value if the peptide p^(k) is associated witha C-terminal flanking sequence that is known to negatively impactpresentation of the peptide p^(k).

According to equation (8), the per-allele likelihood that a peptidesequence p^(k) will be presented by a MHC allele h can be generated byapplying the function g_(h)(⋅) for the MHC allele h to the encodedversion of the peptide sequence p to generate the correspondingdependency score for allele interacting variables. The function g_(w)(⋅)for the allele noninteracting variables are also applied to the encodedversion of the allele noninteracting variables to generate thedependency score for the allele noninteracting variables. Both scoresare combined, and the combined score is transformed by thetransformation function ƒ(⋅) to generate a per-allele likelihood thatthe peptide sequence p^(k) will be presented by the MHC allele h.

Alternatively, the training module 316 may include allele-noninteractingvariables w^(k) in the prediction by adding the allele-noninteractingvariables w^(k) to the allele-interacting variables x_(h) ^(k) inequation (2). Thus, the presentation likelihood can be given by:

u _(k) ^(h) =Pr(p ^(k) presented;allele h)=ƒ(g _(h)([x _(h) ^(k) w^(k)];θ_(h))).  (9)

VIII.B.3 Dependency Functions for Allele-Noninteracting Variables

Similarly to the dependency function g_(h)(⋅) for allele-interactingvariables, the dependency function g_(w)(⋅) for allele noninteractingvariables may be an afTine function or a network function in which aseparate network model is associated with allele-noninteractingvariables w^(k).

Specifically, the dependency function g_(w)(⋅) is an affine functiongiven by:

g _(w)(w ^(k);θ_(w))=w ^(k)·θ_(w).

that linearly combines the allele-noninteracting variables in w^(k) witha corresponding parameter in the set of parameters θ_(w).

The dependency function g_(w)(⋅) may also be a network function givenby:

g _(w)(w ^(k);θ_(w))=NN_(w)(w ^(k);θ_(w)).

represented by a network model NN_(w)(⋅) having an associated parameterin the set of parameters θ_(w). The network function may also includeone or more network models each taking different allele noninteractingvariables as input.

In another instance, the dependency function g_(w)(⋅) for theallele-noninteracting variables can be given by:

g _(w)(w ^(k);θ_(w))=g′ _(w)(w ^(k);θ′_(w))+h(m ^(k);θ_(w) ^(m)),  (10)

where g′_(w)(w^(k):θ′_(w)) is the affine function, the network functionwith the set of allele noninteracting parameters θ′_(w), or the like,m^(k) is the mRNA quantification measurement for peptide p^(k), h(⋅) isa function transforming the quantification measurement, and θ_(w) ^(m)is a parameter in the set of parameters for allele noninteractingvariables that is combined with the mRNA quantification measurement togenerate a dependency score for the mRNA quantification measurement. Inone particular embodiment referred throughout the remainder of thespecification, h(⋅) is the log function, however in practice h(⋅) may beany one of a variety of different functions.

In yet another instance, the dependency function g_(w)(⋅) for theallele-noninteracting variables can be given by:

g _(w)(w ^(k) ;g _(w))=g′ _(w)(w ^(k) ;g′ _(w))+θ_(w) ^(o) ·o^(k),  (11)

where g′_(w)(w^(k);θ′_(w)) is the affine function, the network functionwith the set of allele noninteracting parameters θ′_(w), or the like,o^(k) is the indicator vector described in Section VII.C.2 representingproteins and isoforms in the human proteome for peptide p^(k), and θ_(w)^(o) is a set of parameters in the set of parameters for allelenoninteracting variables that is combined with the indicator vector. Inone variation, when the dimensionality of o^(k) and the set ofparameters ON are significantly high, a parameter regularization term,such as λ·∥θ_(w) ^(o)∥, where ∥⋅∥ represents L1 norm, L2 norm, acombination, or the like, can be added to the loss function whendetermining the value of the parameters. The optimal value of thehyperparameter λ can be determined through appropriate methods.

In yet another instance, the dependency function g_(w)(⋅) for theallele-noninteracting variables can be given by:

$\begin{matrix}{{{g_{w}\left( {w^{k};\theta_{w}} \right)} = {{g_{w}^{\prime}\left( {w^{k};\theta_{w}^{\prime}} \right)} + {\sum\limits_{l = 1}^{L}{\left( {{gene}\left( {p^{k} = l} \right)} \right)\theta_{w}^{l}}}}},} & (12)\end{matrix}$

where g′_(w)(w^(k);θ′_(w)) is the affine function, the network functionwith the set of allele noninteracting parameters θ′_(w), or the like,

(gene(p^(k)=₁)) is the indicator function that equals to 1 if peptidep^(k) is from source gene l as described above in reference to allelenoninteracting variables, and θ_(w) ^(l) is a parameter indicating“antigenicity” of source gene l. In one variation, when L issignificantly high, and thus, the number of parameters θ_(w)^(l=1, 2 . . . , L) are significantly high, a parameter regularizationterm, such as λ·∥θ_(w) ^(l)∥, where ∥⋅∥ represents L1 norm, L2 norm, acombination, or the like, can be added to the loss function whendetermining the value of the parameters. The optimal value of thehyperparameter λ can be determined through appropriate methods.

In yet another instance, the dependency function g_(w)(⋅) for theallele-noninteracting variables can be given by:

$\begin{matrix}{{{g_{w}\left( {w^{k};\theta_{w}} \right)} = {{g_{w}^{\prime}\left( {w^{k};\theta_{w}^{\prime}} \right)} + {\sum\limits_{m = 1}^{M}{\sum\limits_{l = 1}^{L}{{\left( {{{{gene}\left( p^{k} \right)} = l},{{{tissue}\left( p^{k} \right)} = m}} \right) \cdot \theta_{w}^{lm}}}}}}},} & \left( {12b} \right)\end{matrix}$

where g′_(w)(w^(k);θ′_(w)) is the affine function, the network functionwith the set of allele noninteracting parameters θ′_(w), or the like.

(gene(p^(k))=1, tissue(p^(k))=m) is the indicator function that equalsto 1 if peptide p^(k) is from source gene l and if peptide p^(k) is fromtissue type m as described above in reference to allele noninteractingvariables, and θ_(w) ^(lm) is a parameter indicating antigenicity of thecombination of source gene l and tissue type m. Specifically, theantigenicity of gene l for tissue type m may denote the residualpropensity for cells of tissue type m to present peptides from gene Iafter controlling for RNA expression and peptide sequence context.

In one variation, when L or M is significantly high, and thus, thenumber of parameters θ_(w) ^(lm×1, 2, . . . , LM) are significantlyhigh, a parameter regularization term, such as as λ·∥θ_(w) ^(lm)∥, where∥⋅∥ represents L1 norm, L2 norm, a combination, or the like, can beadded to the loss function when determining the value of the parameters.The optimal value of the hyperparameter λ can be determined throughappropriate methods. In another variation, a parameter regularizationterm can be added to the loss function when determining the value of theparameters, such that the coefficients for the same source gene do notsignificantly differ between tissue types. For example, a penalizationterm such as:

$\lambda \cdot {\sum\limits_{l = 1}^{L}\sqrt{\sum\limits_{m = 1}^{M}\left( {\theta_{w}^{l\; m} - \overset{\_}{\theta_{w}^{l}}} \right)^{2}}}$

where θ_(w) ^(l) is the average antigenicity across tissue types forsource gene l, may penalize the standard deviation of antigenicityacross different tissue types in the loss function.

In practice, the additional terms of any of equations (10), (11), (12a)and (12b) may be combined to generate the dependency function g_(w)(⋅)for allele noninteracting variables. For example, the term h(⋅)indicating mRNA quantification measurement in equation (10) and the termindicating source gene antigenicity in equation (12) may be summedtogether along with any other affine or network function to generate thedependency function for allele noninteracting variables.

Returning to equation (8), as an example, the likelihood that peptidep^(k) will be presented by MHC allele h=3, among m=4 differentidentified MHC alleles using the affine transformation functionsg_(h)(⋅), g_(w)(⋅), can be generated by:

u _(k) ³=ƒ(w ^(k)·θ_(w) +x ₃ ^(k)·θ₃),

where w^(k) are the identified allele-noninteracting variables forpeptide p^(k), and θ_(w) are the set of parameters determined for theallele-noninteracting variables.

As another example, the likelihood that peptide p^(k) will be presentedby MHC allele h=3, among m=4 different identified MHC alleles using thenetwork transformation functions g_(h)(⋅), g_(w)(ω), can be generatedby:

u _(k) ³=ƒ(NN_(w)(w ^(k);θ_(w))+NN₃(x ₃ ^(k);θ₃))

where w^(k) are the identified allele-interacting variables for peptidep^(k), and θ_(w) are the set of parameters determined forallele-noninteracting variables.

FIG. 8 illustrates generating a presentation likelihood for peptidep^(k) in association with MHC allele h=3 using example network modelsNN₃(⋅) and NN_(w)(⋅). As shown in FIG. 8, the network model NN₃(⋅)receives the allele-interacting variables x₃ ^(k) for MHC allele h=3 andgenerates the output NN₃(x₃ ^(k)). The network model NN_(w)(⋅) receivesthe allele-noninteracting variables w^(k) for peptide p^(k) andgenerates the output NN_(w)(w^(k)). The outputs are combined and mappedby function ƒ(⋅) to generate the estimated presentation likelihood u.

VIII.C. Multiple-Allele Models

The training module 316 may also construct the presentation models topredict presentation likelihoods of peptides in a multiple-allelesetting where two or more MHC alleles are present. In this case, thetraining module 316 may train the presentation models based on datainstances S in the training data 170 generated from cells expressingsingle MHC alleles, cells expressing multiple MHC alleles, or acombination thereof.

VIII.C.1. Example 1: Maximum of Per-Allele Models

In one implementation, the training module 316 models the estimatedpresentation likelihood u_(k) for peptide p^(k) in association with aset of multiple MHC alleles H as a function of the presentationlikelihoods u_(k) ^(h) ^(∈H) determined for each of the MHC alleles h inthe set H determined based on cells expressing single-alleles, asdescribed above in conjunction with equations (2)-(11). Specifically,the presentation likelihood u_(k) can be any function of u_(k) ^(h∈H).In one implementation, as shown in equation (12), the function is themaximum function, and the presentation likelihood u_(k) can bedetermined as the maximum of the presentation likelihoods for each MHCallele h in the set H.

u _(k) =Pr(p ^(k) presented;allelesH)=max(u _(k) ^(h∈N)).

VIIi.C.2. Example 2.1: Function-of-Sums Models

In one implementation, the training module 316 models the estimatedpresentation likelihood u_(k) for peptide p^(k) by:

$\begin{matrix}{{u_{k} = {{\Pr \left( {p^{k}\mspace{14mu} {presented}} \right)} = {f\left( {\sum\limits_{h = 1}^{m}{a_{h}^{k} \cdot {g_{h}\left( {x_{h}^{k};\theta_{h}} \right)}}} \right)}}},} & (13)\end{matrix}$

where elements a_(h) ^(k) are 1 for the multiple MHC alleles Hassociated with peptide sequence p^(k) and x_(h) ^(k) denotes theencoded allele-interacting variables for peptide p^(k) and thecorresponding MHC alleles. The values for the set of parameters θ_(k)for each MHC allele h can be determined by minimizing the loss functionwith respect to θ_(h), where i is each instance in the subset S oftraining data 170 generated from cells expressing single MHC allelesand/or cells expressing multiple MHC alleles. The dependency functiong_(h) may be in the form of any of the dependency functions g_(h)introduced above in sections VIII.B.1.

According to equation (13), the presentation likelihood that a peptidesequence p^(k) will be presented by one or more MHC alleles h can begenerated by applying the dependency function g_(h)(⋅) to the encodedversion of the peptide sequence p^(k) for each of the MHC alleles H togenerate the corresponding score for the allele interacting variables.The scores for each MHC allele h are combined, and transformed by thetransformation function ƒ(⋅) to generate the presentation likelihoodthat peptide sequence p^(k) will be presented by the set of MHC allelesH.

The presentation model of equation (13) is different from the per-allelemodel of equation (2), in that the number of associated alleles for eachpeptide p^(k) can be greater than 1. In other words, more than oneelement in a_(h) ^(k) can have values of 1 for the multiple MHC allelesH associated with peptide sequence p^(k).

As an example, the likelihood that peptide p^(k) will be presented byMHC alleles h=2, h=3, among m=4 different identified MHC alleles usingthe affine transformation functions g_(h)(⋅), can be generated by:

u _(k)=ƒ(x ₂ ^(k)·θ₂ +x ₃ ^(k)·θ₃),

where x₂ ^(k), x₃ ^(k) are the identified allele-interacting variablesfor MHC alleles h=2, h=3, and θ₂, θ₃ are the set of parametersdetermined for MHC alleles h=2, h=3.

As another example, the likelihood that peptide p^(k) will be presentedby MHC alleles h=2, h=3, among m=4 different identified MHC allelesusing the network transformation functions g_(h)(⋅), g_(w)(⋅), can begenerated by:

u _(k)=ƒ(NN₂(x ₂ ^(k);θ₂)+NN₃(x ₃ ^(k);θ₃)),

where NN₂(⋅), NN₃(⋅) are the identified network models for MHC allelesh=2, h=3, and θ₂, θ₃ are the set of parameters determined for MHCalleles h=2, h=3.

FIG. 9 illustrates generating a presentation likelihood for peptidep^(k) in association with MHC alleles h=2, h=3 using example networkmodels NN₂(⋅) and NN₃(⋅). As shown in FIG. 9, the network model NN₂(⋅)receives the allele-interacting variables x₂ for MHC allele h=2 andgenerates the output NN₂(x₂ ^(k)) and the network model NN₃(⋅) receivesthe allele-interacting variables x₃ ^(k) for MHC allele h=3 andgenerates the output NN₃(x₃ ^(k)). The outputs are combined and mappedby function ƒ(⋅) to generate the estimated presentation likelihoodu_(k).

VIII.C.3. Example 2.2: Function-of-Sums Models withAllele-Noninteracting Variables

In one implementation, the training module 316 incorporatesallele-noninteracting variables and models the estimated presentationlikelihood u_(k) for peptide p^(k) by:

$\begin{matrix}{{u_{k} = {{\Pr \left( {p^{k}\mspace{14mu} {presented}} \right)} = {f\left( {{g_{w}\left( {w^{k};\theta_{w}} \right)} + {\sum\limits_{h = 1}^{m}{a_{h}^{k} \cdot {g_{h}\left( {x_{h}^{k};\theta_{h}} \right)}}}} \right)}}},} & (14)\end{matrix}$

where w^(k) denotes the encoded allele-noninteracting variables forpeptide p^(k). Specifically, the values for the set of parameters θ_(h)for each MHC allele h and the set of parameters θ_(w) forallele-noninteracting variables can be determined by minimizing the lossfunction with respect to θ_(h) and θ_(w), where i is each instance inthe subset S of training data 170 generated from cells expressing singleMHC alleles and/or cells expressing multiple MHC alleles. The dependencyfunction g_(w) may be in the form of any of the dependency functionsg_(w) introduced above in sections VIII.B.3.

Thus, according to equation (14), the presentation likelihood that apeptide sequence p will be presented by one or more MHC alleles H can begenerated by applying the function g_(h)(⋅) to the encoded version ofthe peptide sequence p^(k) for each of the MHC alleles H to generate thecorresponding dependency score for allele interacting variables for eachMHC allele h. The function g_(w)(⋅) for the allele noninteractingvariables is also applied to the encoded version of the allelenoninteracting variables to generate the dependency score for the allelenoninteracting variables. The scores are combined, and the combinedscore is transformed by the transformation function ƒ(⋅) to generate thepresentation likelihood that peptide sequence p^(k) will be presented bythe MHC alleles H.

In the presentation model of equation (14), the number of associatedalleles for each peptide p^(k) can be greater than 1. In other words,more than one element in a_(h) ^(k) can have values of 1 for themultiple MHC alleles H associated with peptide sequence p^(k).

As an example, the likelihood that peptide p^(k) will be presented byMHC alleles h=2, h=3, among m=4 different identified MHC alleles usingthe affine transformation functions g_(h)(⋅), g_(w)(⋅), can be generatedby:

u _(k)=ƒ(w ^(k)·θ_(w) +x ₂ ^(k)·θ₂ +x ₃ ^(k)·θ₃),

where i are the identified allele-noninteracting variables for peptidep^(k), and θ_(w) are the set of parameters determined for theallele-noninteracting variables.

As another example, the likelihood that peptide p^(k) will be presentedby MHC alleles h=2, h=3, among m=4 different identified MHC allelesusing the network transformation functions g_(h)(⋅), g_(w)(⋅), can begenerated by:

u _(k)=ƒ(NN_(w)(w ^(k);θ_(w))+NN₂(x ₂ ^(k);θ₂)+NN₃(x ₃ ^(k);θ₃))

where w^(k) are the identified allele-interacting variables for peptidep^(k), and θ_(w) are the set of parameters determined forallele-noninteracting variables.

FIG. 10 illustrates generating a presentation likelihood for peptidep^(k) in association with MHC alleles h=2, h=3 using example networkmodels NN₂(⋅), NN₃(⋅), and NN_(w)(⋅). As shown in FIG. 10, the networkmodel NN₂(⋅) receives the allele-interacting variables x₂ ^(k) for MHCallele h=2 and generates the output NN₂(x₂ ^(k)). The network modelNN₃(⋅) receives the allele-interacting variables x₃ ^(k) for MHC alleleh=3 and generates the output NN₃(x₃ ^(k)). The network model NN_(w)(⋅)receives the allele-noninteracting variables w^(k) for peptide p^(k) andgenerates the output NN_(w)(w^(k)). The outputs are combined and mappedby function ƒ(⋅) to generate the estimated presentation likelihoodu_(k).

Alternatively, the training module 316 may include allele-noninteractingvariables w^(k) in the prediction by adding the allele-noninteractingvariables w^(k) to the allele-interacting variables x_(h) ^(k) inequation (15). Thus, the presentation likelihood can be given by:

$\begin{matrix}{u_{\kappa} = {{\Pr \left( {p^{k}\mspace{14mu} {presented}} \right)} = {{f\left( {\sum\limits_{h = 1}^{m}{a_{h}^{k} \cdot {g_{h}\left( {\left\lbrack {x_{h}^{k}w^{k}} \right\rbrack;\theta_{h}} \right)}}} \right)}.}}} & (15)\end{matrix}$

VIII.C.4. Example 3.1: Models Using Implicit Per-Allele Likelihoods

In another implementation, the training module 316 models the estimatedpresentation likelihood u_(k) for peptide p^(k) by:

u _(k) =Pr(p ^(k) presented)=r(s(v=[a ₁ ^(k) ·u′ _(k) ¹(θ) . . . a _(m)^(k) ·u′ _(k) ^(m)(θ)])),  (16)

where elements a_(h) ^(k) are 1 for the multiple MHC alleles h∈Hassociated with peptide sequence p^(k), u′_(k) ^(h) is an implicitper-allele presentation likelihood for MHC allele h, vector v is avector in which element v_(h) corresponds to a_(h) ^(k)·u′_(k) ^(h),s(⋅) is a function mapping the elements of v, and r(⋅) is a clippingfunction that clips the value of the input into a given range. Asdescribed below in more detail, s(⋅) may be the summation function orthe second-order function, but it is appreciated that in otherembodiments, s(⋅) can be any function such as the maximum function. Thevalues for the set of parameters θ for the implicit per-allelelikelihoods can be determined by minimizing the loss function withrespect to θ, where i is each instance in the subset S of training data170 generated from cells expressing single MHC alleles and/or cellsexpressing multiple MHC alleles.

The presentation likelihood in the presentation model of equation (17)is modeled as a function of implicit per-allele presentation likelihoodsu′_(k) ^(h) that each correspond to the likelihood peptide p^(k) will bepresented by an individual MHC allele h. The implicit per-allelelikelihood is distinct from the per-allele presentation likelihood ofsection VIII.B in that the parameters for implicit per-allelelikelihoods can be learned from multiple allele settings, in whichdirect association between a presented peptide and the corresponding MHCallele is unknown, in addition to single-allele settings. Thus, in amultiple-allele setting, the presentation model can estimate not onlywhether peptide p^(k) will be presented by a set of MHC alleles H as awhole, but can also provide individual likelihoods u′_(k) ^(h) ^(∈H)that indicate which MHC allele h most likely presented peptide p^(k). Anadvantage of this is that the presentation model can generate theimplicit likelihoods without training data for cells expressing singleMHC alleles.

In one particular implementation referred throughout the remainder ofthe specification, r(⋅) is a function having the range [0, 1]. Forexample, r(⋅) may be the clip function:

r(z)=min(max(z,0),1),

where the minimum value between z and 1 is chosen as the presentationlikelihood u_(k). In another implementation, r(⋅) is the hyperbolictangent function given by:

r(z)=tanh(z)

when the values for the domain z is equal to or greater than 0.

VIII.C.5. Example 3.2: Sum-of-Functions Model

In one particular implementation, s(⋅) is a summation function, and thepresentation likelihood is given by summing the implicit per-allelepresentation likelihoods:

$\begin{matrix}{u_{k} = {{\Pr \left( {p^{k}\mspace{14mu} {presented}} \right)} = {{r\left( {\sum\limits_{h = 1}^{m}{a_{h}^{k} \cdot {u_{k}^{\prime \; h}(\theta)}}} \right)}.}}} & (17)\end{matrix}$

In one implementation, the implicit per-allele presentation likelihoodfor MHC allele h is generated by:

u′ _(k) ^(h)=ƒ(g _(h)(x _(h) ^(k);θ_(h))),  (18)

such that the presentation likelihood is estimated by:

$\begin{matrix}{u_{k} = {{\Pr \left( {p^{k}\mspace{14mu} {presented}} \right)} = {{r\left( {\sum\limits_{h = 1}^{m}{a_{h}^{k} \cdot {f\left( {g_{h}\left( {x_{h}^{k};\theta_{h}} \right)} \right)}}} \right)}.}}} & (19)\end{matrix}$

According to equation (19), the presentation likelihood that a peptidesequence p^(k) will be presented by one or more MHC alleles H can begenerated by applying the function g_(h)(⋅) to the encoded version ofthe peptide sequence p^(k) for each of the MHC alleles H to generate thecorresponding dependency score for allele interacting variables. Eachdependency score is first transformed by the function ƒ(⋅) to generateimplicit per-allele presentation likelihoods u′_(k) ^(h). The per-allelelikelihoods u′_(k) ^(h) are combined, and the clipping function may beapplied to the combined likelihoods to clip the values into a range [0,1] to generate the presentation likelihood that peptide sequence p^(k)will be presented by the set of MHC alleles H. The dependency functiong_(h) may be in the form of any of the dependency functions g_(h)introduced above in sections VIII.B.1.

As an example, the likelihood that peptide p^(k) will be presented byMHC alleles h=2, h=3, among m=4 different identified MHC alleles usingthe affine transformation functions g_(h)(⋅), can be generated by:

u _(k) =r(ƒ(x ₂ ^(k)·θ₂)+ƒ(x ₃ ^(k)·θ₃)),

where x₂ ^(k), x₃ ^(k) are the identified allele-interacting variablesfor MHC alleles h=2, h=3, and θ₂, θ₃ are the set of parametersdetermined for MHC alleles h=2, h=3.

As another example, the likelihood that peptide p^(k) will be presentedby MHC alleles h=2, h=3, among m=4 different identified MHC allelesusing the network transformation functions g_(h)(⋅), g_(w)(⋅), can begenerated by:

u _(k) =r(ƒ(NN₂(x ₂ ^(k);θ₂))+ƒ(NN₃(x ₃ ^(k);θ₃))),

where NN₂(⋅), NN₃(⋅) are the identified network models for MHC allelesh=2, h=3, and θ₂, θ₃ are the set of parameters determined for MHCalleles h=2, h=3.

FIG. 11 illustrates generating a presentation likelihood for peptidep^(k) in association with MHC alleles h=2, h=3 using example networkmodels NN₂(⋅) and NN₃(⋅). As shown in FIG. 9, the network model NN₂(⋅)receives the allele-interacting variables x₂ ^(k) for MHC allele h=2 andgenerates the output NN₂(x₂ ^(k)) and the network model NN₃(⋅) receivesthe allele-interacting variables x₃ for MHC allele h=3 and generates theoutput NN₃(x₃ ^(k)). Each output is mapped by function ƒ(⋅) and combinedto generate the estimated presentation likelihood u_(k).

In another implementation, when the predictions are made for the log ofmass spectrometry ion currents, r(⋅) is the log function and ƒ(⋅) is theexponential function.

VIII.C.6. Example 3.3: Sum-of-Functions Models withAllele-Noninteracting Variables

In one implementation, the implicit per-allele presentation likelihoodfor MHC allele h is generated by:

u′ _(k) ^(h)=ƒ(g _(h)(x _(h) ^(k);θ_(h))+g _(w)(w ^(k);θ_(w))),  (20)

such that the presentation likelihood is generated by:

$\begin{matrix}{{u_{k} = {{\Pr \left( {p^{k}\mspace{14mu} {presented}} \right)} = {r\left( {\sum\limits_{h = 1}^{m}{a_{h}^{k} \cdot {f\left( {{g_{w}\left( {w^{l};\theta_{w}} \right)} + {g_{h}\left( {x_{h}^{k};\theta_{h}} \right)}} \right)}}} \right)}}},} & (21)\end{matrix}$

to incorporate the impact of allele noninteracting variables on peptidepresentation.

According to equation (21), the presentation likelihood that a peptidesequence p^(k) will be presented by one or more MHC alleles H can begenerated by applying the function g_(h)(⋅) to the encoded version ofthe peptide sequence p^(k) for each of the MHC alleles H to generate thecorresponding dependency score for allele interacting variables for eachMHC allele h. The function g_(w)(⋅) for the allele noninteractingvariables is also applied to the encoded version of the allelenoninteracting variables to generate the dependency score for the allelenoninteracting variables. The score for the allele noninteractingvariables are combined to each of the dependency scores for the alleleinteracting variables. Each of the combined scores are transformed bythe function ƒ(⋅) to generate the implicit per-allele presentationlikelihoods. The implicit likelihoods are combined, and the clippingfunction may be applied to the combined outputs to clip the values intoa range [0,1] to generate the presentation likelihood that peptidesequence p^(k) will be presented by the MHC alleles H. The dependencyfunction g_(w) may be in the form of any of the dependency functionsg_(w) introduced above in sections VIII.B.3.

As an example, the likelihood that peptide p^(k) will be presented byMHC alleles h=2, h=3, among m=4 different identified MHC alleles usingthe affine transformation functions g_(h)(⋅), g_(w)(⋅), can be generatedby:

u _(k) =r(ƒ(w ^(k)·θ_(w) +x ₂ ^(k)·θ₂)+ƒ(w ^(k)·θ_(w) +x ₃ ^(k)·θ₃)),

where w^(k) are the identified allele-noninteracting variables forpeptide p^(k), and θ_(w) are the set of parameters determined for theallele-noninteracting variables.

As another example, the likelihood that peptide p^(k) will be presentedby MHC alleles h=2, h=3, among m=4 different identified MHC allelesusing the network transformation functions g_(h)(⋅), g_(w)(⋅), can begenerated by:

u _(k) =r(ƒ(NN_(w)(w ^(k);θ_(w))+NN₂(x ₂ ^(k);θ₂))+ƒ(NN_(w)(w^(k);θ_(w))+NN₃(x ₃ ^(k);θ₃)))

where w^(k) are the identified allele-interacting variables for peptidep^(k), and θ_(w) are the set of parameters determined forallele-noninteracting variables.

FIG. 12 illustrates generating a presentation likelihood for peptidep^(k) in association with MHC alleles h=2, h=3 using example networkmodels NN₂(⋅), NN₃(⋅), and NN_(w)(⋅). As shown in FIG. 12, the networkmodel NN₂(⋅) receives the allele-interacting variables x₂ ^(k) for MHCallele h=2 and generates the output NN₂(x₂ ^(k)). The network modelNN_(w)(⋅) receives the allele-noninteracting variables w^(k) for peptidep^(k) and generates the output NN_(w)(w^(k)). The outputs are combinedand mapped by function ƒ(⋅). The network model NN₃(⋅) receives theallele-interacting variables x₃ ^(k) for MHC allele h=3 and generatesthe output NN₃(x₃ ^(k)), which is again combined with the outputNN_(w)(w^(k)) of the same network model NN_(w)(⋅) and mapped by functionƒ(⋅). Both outputs are combined to generate the estimated presentationlikelihood u_(k).

In another implementation, the implicit per-allele presentationlikelihood for MHC allele h is generated by:

u′ _(k) ^(h)=ƒ(g _(h)([x _(h) ^(k) w ^(k) k];θ_(h)))  (22)

such that the presentation likelihood is generated by:

$u_{k} = {{\Pr \left( {p^{k}\mspace{14mu} {presented}} \right)} = {{r\left( {\sum\limits_{h = 1}^{m}{a_{h}^{k} \cdot {f\left( {g_{h}\left( {\left\lbrack {x_{h}^{k}w^{k}} \right\rbrack;\theta_{h}} \right)} \right)}}} \right)}.}}$

VIII.C.7. Example 4: Second Order Models

In one implementation, s(⋅) is a second-order function, and theestimated presentation likelihood u_(k) for peptide p^(k) is given by:

$\begin{matrix}{u_{k} = {{\Pr \left( {p^{k}\mspace{14mu} {presented}} \right)} = {{\sum\limits_{h = 1}^{m}{a_{h}^{k} \cdot {u_{k}^{\prime \; h}(\theta)}}} - {\sum\limits_{h = 1}^{m}{\sum\limits_{j < h}{a_{h}^{k} \cdot a_{j}^{k} \cdot {u_{k}^{\prime \; h}(\theta)} \cdot {u_{k}^{\prime \; j}(\theta)}}}}}}} & (23)\end{matrix}$

where elements u′_(k) ^(h) are the implicit per-allele presentationlikelihood for MHC allele h. The values for the set of parameters θ forthe implicit per-allele likelihoods can be determined by minimizing theloss function with respect to θ, where i is each instance in the subsetS of training data 170 generated from cells expressing single MHCalleles and/or cells expressing multiple MHC alleles. The implicitper-allele presentation likelihoods may be in any form shown inequations (18), (20), and (22) described above.

In one aspect, the model of equation (23) may imply that there exists apossibility peptide p^(k) will be presented by two MHC allelessimultaneously, in which the presentation by two HLA alleles isstatistically independent.

According to equation (23), the presentation likelihood that a peptidesequence p^(k) will be presented by one or more MHC alleles H can begenerated by combining the implicit per-allele presentation likelihoodsand subtracting the likelihood that each pair of MHC alleles willsimultaneously present the peptide p^(k) from the summation to generatethe presentation likelihood that peptide sequence p^(k) will bepresented by the MHC alleles H.

As an example, the likelihood that peptide p^(k) will be presented byHLA alleles h=2, h=3, among m=4 different identified HLA alleles usingthe affine transformation functions g_(h)(⋅), can be generated by:

u _(k)=ƒ(x ₂ ^(k)·θ₂)+ƒ(x ₃ ^(k)·θ₃)−ƒ(x ₂ ^(k)·θ₂)·ƒ(x ₂ ^(k)·θ₂),

where x₂ ^(k), x₃ ^(k) are the identified allele-interacting variablesfor HLA alleles h=2, h=3, and θ₂, θ₃ are the set of parametersdetermined for HLA alleles h=2, h=3.

As another example, the likelihood that peptide p^(k) will be presentedby HLA alleles h=2, h=3, among m=4 different identified HLA allelesusing the network transformation functions g_(h)(⋅), g_(w)(⋅), can begenerated by:

u _(k)=ƒ(NN₂(x ₂ ^(k);θ₂))+ƒ(NN₃(x ₃ ^(k);θ₃))−ƒ(NN₂(x ₂^(k);θ₂))·ƒ(NN₃(x ₃ ^(k);θ₃)),

where NN₂(⋅), NN₃(⋅) are the identified network models for HLA allelesh=2, h=3, and θ₂, θ₃ are the set of parameters determined for HLAalleles h=2, h=3.

IX. Example 5: Prediction Module

The prediction module 320 receives sequence data and selects candidateneoantigens in the sequence data using the presentation models.Specifically, the sequence data may be DNA sequences, RNA sequences,and/or protein sequences extracted from tumor tissue cells of patients.The prediction module 320 processes the sequence data into a pluralityof peptide sequences p^(k) having 8-15 amino acids for MHC-I or 6-30amino acids for MHC-II. For example, the prediction module 320 mayprocess the given sequence “IEFROEIFJEF (SEQ ID NO: 16) into threepeptide sequences having 9 amino acids “IEFROEIFJ (SEQ ID NO: 17),”“EFROEIFJE (SEQ ID NO: 18),” and “FROEIFJEF (SEQ ID NO: 19).” In oneembodiment, the prediction module 320 may identify candidate neoantigensthat are mutated peptide sequences by comparing sequence data extractedfrom normal tissue cells of a patient with the sequence data extractedfrom tumor tissue cells of the patient to identify portions containingone or more mutations.

The prediction module 320 applies one or more of the presentation modelsto the processed peptide sequences to estimate presentation likelihoodsof the peptide sequences. Specifically, the prediction module 320 mayselect one or more candidate neoantigen peptide sequences that arelikely to be presented on tumor HLA molecules by applying thepresentation models to the candidate neoantigens. In one implementation,the prediction module 320 selects candidate neoantigen sequences thathave estimated presentation likelihoods above a predetermined threshold.In another implementation, the presentation model selects the νcandidate neoantigen sequences that have the highest estimatedpresentation likelihoods (where ν is generally the maximum number ofepitopes that can be delivered in a vaccine). A vaccine including theselected candidate neoantigens for a given patient can be injected intothe patient to induce immune responses.

X. Example 6: Patient Selection Module

The patient selection module 324 selects a subset of patients forvaccine treatment and/or T-cell therapy based on whether the patientssatisfy inclusion criteria. In one embodiment, the inclusion criteria isdetermined based on the presentation likelihoods of patient neoantigencandidates as generated by the presentation models. By adjusting theinclusion criteria, the patient selection module 324 can adjust thenumber of patients that will receive the vaccine and/or T-cell therapybased on his or her presentation likelihoods of neoantigen candidates.Specifically, a stringent inclusion criteria results in a fewer numberof patients that will be treated with the vaccine and/or T-cell therapy,but may result in a higher proportion of vaccine and/or T-celltherapy-treated patients that receive effective treatment (e.g., 1 ormore tumor-specific neoantigens (TSNA) and/or 1 or moreneoantigen-responsive T-cells). On the other hand, a lenient inclusioncriteria results in a higher number of patients that will be treatedwith the vaccine and/or with T-cell therapy, but may result in a lowerproportion of vaccine and/or T-cell therapy-treated patients thatreceive effective treatment. The patient selection module 324 modifiesthe inclusion criteria based on the desired balance between targetproportion of patients that will receive treatment and proportion ofpatients that receive effective treatment.

In some embodiments, inclusion criteria for selection of patients toreceive vaccine treatment are the same as inclusion criteria forselection of patients to receive T-cell therapy. However, in alternativeembodiments, inclusion criteria for selection of patients to receivevaccine treatment may differ from inclusion criteria for selection ofpatients to receive T-cell therapy. The following Sections X.A and X.Bdiscuss inclusion criteria for selection of patients to receive vaccinetreatment and inclusion criteria for selection of patients to receiveT-cell therapy, respectively.

X.A. Patient Selection for Vaccine Treatment

In one embodiment, patients are associated with a correspondingtreatment subset of v neoantigen candidates that can potentially beincluded in customized vaccines for the patients with vaccine capacityv. In one embodiment, the treatment subset for a patient are theneoantigen candidates with the highest presentation likelihoods asdetermined by the presentation models. For example, if a vaccine caninclude v=20 epitopes, the vaccine can include the treatment subset ofeach patient that have the highest presentation likelihoods asdetermined by the presentation model. However, it is appreciated that inother embodiments, the treatment subset for a patient can be determinedbased on other methods. For example, the treatment subset for a patientmay be randomly selected from the set of neoantigen candidates for thepatient, or may be determined in part based on current state-of-the-artmodels that model binding affinity or stability of peptide sequences, orsome combination of factors that include presentation likelihoods fromthe presentation models and affinity or stability information regardingthose peptide sequences.

In one embodiment, the patient selection module 324 determines that apatient satisfies the inclusion criteria if the tumor mutation burden ofthe patient is equal to or above a minimum mutation burden. The tumormutation burden (TMB) of a patient indicates the total number ofnonsynonymous mutations in the tumor exome. In one implementation, thepatient selection module 324 may select a patient for vaccine treatmentif the absolute number of TMB of the patient is equal to or above apredetermined threshold. In another implementation, the patientselection module 324 may select a patient for vaccine treatment if theTMB of the patient is within a threshold percentile among the TMB'sdetermined for the set of patients.

In another embodiment, the patient selection module 324 determines thata patient satisfies the inclusion criteria if a utility score of thepatient based on the treatment subset of the patient is equal to orabove a minimum utility score. In one implementation, the utility scoreis a measure of the estimated number of presented neoantigens from thetreatment subset.

The estimated number of presented neoantigens may be predicted bymodeling neoantigen presentation as a random variable of one or moreprobability distributions. In one implementation, the utility score forpatient i is the expected number of presented neoantigen candidates fromthe treatment subset, or some function thereof. As an example, thepresentation of each neoantigen can be modeled as a Bemoulli randomvariable, in which the probability of presentation (success) is given bythe presentation likelihood of the neoantigen candidate. Specifically,for a treatment subset S_(i) of v neoantigen candidates p^(i1), p^(i2),. . . , p^(iv) each having the highest presentation likelihoods u_(i1),u_(i2), . . . , u_(iv), presentation of neoantigen candidate p^(ij) isgiven by random variable A_(ij), in which:

P(A _(ij)=1)=u _(ij) ,P(A _(ij)=0)=1−u _(ij).  (24)

The expected number of presented neoantigens is given by the summationof the presentation likelihoods for each neoantigen candidate. In otherwords, the utility score for patient i can be expressed as:

$\begin{matrix}{{uti{l_{i}\left( S_{i} \right)}} = {{\left\lbrack {\sum\limits_{j = 1}^{v}A_{ij}} \right\rbrack} = {\sum\limits_{j = 1}^{v}{u_{ij}.}}}} & (25)\end{matrix}$

The patient selection module 324 selects a subset of patients havingutility scores equal to or above a minimum utility for vaccinetreatment.

In another implementation, the utility score for patient i is theprobability that at least a threshold number of neoantigens k will bepresented. In one instance, the number of presented neoantigens in thetreatment subset S_(i) of neoantigen candidates is modeled as a PoissonBinomial random variable, in which the probabilities of presentation(successes) are given by the presentation likelihoods of each of theepitopes. Specifically, the number of presented neoantigens for patienti can be given by random variable N_(i), in which:

$\begin{matrix}{N_{i} = {{\sum\limits_{j = 1}^{v}A_{ij}} \sim {{{PBD}\left( {u_{i\; 1},u_{i2},\ldots \mspace{14mu},u_{iv}} \right)}.}}} & (26)\end{matrix}$

where PBD(⋅) denotes the Poisson Binomial distribution. The probabilitythat at least a threshold number of neoantigens k will be presented isgiven by the summation of the probabilities that the number of presentedneoantigens N_(i) will be equal to or above k. In other words, theutility score for patient i can be expressed as:

$\begin{matrix}{{{util}_{i}\left( S_{i} \right)} = {{{\mathbb{P}}\left\lbrack {N_{i} \geq k} \right\rbrack} = {\sum\limits_{m = 1}^{k}{{{\mathbb{P}}\left\lbrack {N_{i} = m} \right\rbrack}.}}}} & (27)\end{matrix}$

The patient selection module 324 selects a subset of patients having theutility score equal to or above a minimum utility for vaccine treatment.

In another implementation, the utility score for patient i is the numberof neoantigens in the treatment subset S; of neoantigen candidateshaving binding affinity or predicted binding affinity below a fixedthreshold (e.g., 500 nM) to one or more of the patient's HLA alleles. Inone instance, the fixed threshold is a range from 1000 nM to 10 nM.Optionally, the utility score may count only those neoantigens detectedas expressed via RNA-seq.

In another implementation, the utility score for patient i is the numberof neoantigens in the treatment subset S_(i) of neoantigen candidateshaving binding affinity to one or more of that patient's HLA alleles ator below a threshold percentile of binding affinities for randompeptides to that HLA allele. In one instance, the threshold percentileis a range from the 10^(th) percentile to the 0.1^(th) percentile.Optionally, the utility score may count only those neoantigens detectedas expressed via RNA-seq.

It is appreciated that the examples of generating utility scoresillustrated with respect to equations (25) and (27) are merelyillustrative, and the patient selection module 324 may use otherstatistics or probability distributions to generate the utility scores.

X.B. Patient Selection for T-Cell Therapy

In another embodiment, instead of or in addition to receiving vaccinetreatment, patients can receive T-cell therapy. Like vaccine treatment,in embodiments in which a patient receives T-cell therapy, the patientmay be associated with a corresponding treatment subset of v neoantigencandidates as described above. This treatment subset of v neoantigencandidates can be used for in vitro identification of T cells from thepatient that are responsive to one or more of the v neoantigencandidates. These identified T cells can then be expanded and infusedinto the patient for customized T-cell therapy.

Patients may be selected to receive T-cell therapy at two different timepoints. The first point is after the treatment subset of v neoantigencandidates have been predicted for a patient using the models, butbefore in vitro screening for T cells that are specific to the predictedtreatment subset of v neoantigen candidates. The second point is afterin vitro screening for T cells that are specific to the predictedtreatment subset of v neoantigen candidates.

First, patients may be selected to receive T-cell therapy after thetreatment subset of v neoantigen candidates have been predicted for thepatient, but before in vitro identification of T-cells from the patientthat are specific to the predicted subset of v neoantigen candidates.Specifically, because in vitro screening for neoantigen-specific T-cellsfrom the patient can be expensive, it may be desirable to only selectpatients to screen for neoantigen-specific T-cells if the patients arelikely to have neoantigen-specific T-cells. To select patients beforethe in vitro T-cell screening step, the same criteria that are used toselect patients for vaccine treatment may be used. Specifically, in someembodiments, the patient selection module 324 may select a patient toreceive T-cell therapy if the tumor mutation burden of the patient isequal to or above a minimum mutation burden as described above. Inanother embodiment, the patient selection module 324 may select apatient to receive T-cell therapy if a utility score of the patientbased on the treatment subset of v neoantigen candidates for the patientis equal to or above a minimum utility score, as described above.

Second, in addition to or instead of selecting patients to receiveT-cell therapy before in vitro identification of T-cells from thepatient that are specific to the predicted subset of v neoantigencandidates, patients may also be selected to receive T-cell therapyafter in vitro identification of T-cells that are specific to thepredicted treatment subset of v neoantigen candidates. Specifically, apatient may be selected to receive T-cell therapy if at least athreshold quantity of neoantigen-specific TCRs are identified for thepatient during the in vitro screening of the patient's T-cells forneoantigen recognition. For example, a patient may be selected toreceive T-cell therapy only if at least two neoantigen-specific TCRs areidentified for the patient, or only if neoantigen-specific TCRs areidentified for two distinct neoantigens.

In another embodiment, a patient may be selected to receive T-celltherapy only if at least a threshold quantity of neoantigens of thetreatment subset of v neoantigen candidates for the patient arerecognized by the patient's TCRs. For example, a patient may be selectedto receive T-cell therapy only if at least one neoantigen of thetreatment subset of v neoantigen candidates for the patient arerecognized by the patient's TCRs. In further embodiments, a patient maybe selected to receive T-cell therapy only if at least a thresholdquantity of TCRs for the patient are identified as neoantigen-specificto neoantigen peptides of a particular HLA restriction class. Forexample, a patient may be selected to receive T-cell therapy only if atleast one TCR for the patient is identified as neoantigen-specific HLAclass I restricted neoantigen peptides.

In even further embodiments, a patient may be selected to receive T-celltherapy only if at least a threshold quantity of neoantigen peptides ofa particular HLA restriction class are recognized by the patient's TCRs.For example, a patient may be selected to receive T-cell therapy only ifat least one HLA class I restricted neoantigen peptide is recognized bythe patient's TCRs. As another example, a patient may be selected toreceive T-cell therapy only if at least two HLA class II restrictedneoantigen peptides are recognized by the patient's TCRs. Anycombination of the above criteria may also be used for selectingpatients to receive T-cell therapy after in vitro identification ofT-cells that are specific to the predicted treatment subset of vneoantigen candidates for the patient.

XI. Example 7: Experimentation Results Showing Example Patient SelectionPerformance

The validity of patient selection methods described in Section X aretested by performing patient selection on a set of simulated patientseach associated with a test set of simulated neoantigen candidates, inwhich a subset of simulated neoantigens is known to be presented in massspectrometry data. Specifically, each simulated neoantigen candidate inthe test set is associated with a label indicating whether theneoantigen was presented in a multiple-allele JY cell line HLA-A*02:01and HLA-B*07:02 mass spectrometry data set from the Bassani-Stembergdata set (data set “D1”) (data can be found atwww.ebi.ac.u_(k)/pride/archive/projects/PXD0000394). As described inmore detail below in conjunction with FIG. 13A, a number of neoantigencandidates for the simulated patients are sampled from the humanproteome based on the known frequency distribution of mutation burden innon-small cell lung cancer (NSCLC) patients.

Per-allele presentation models for the same HLA alleles are trainedusing a training set that is a subset of the single-allele HLA-A*02:01and HLA-B*07:02 mass spectrometry data from the IEDB data set (data set“D2”) (data can be found athttp://www.iedb.org/doc/mhc_ligand_full.zip). Specifically, thepresentation model for each allele was the per-allele model shown inequation (8) that incorporated N-terminal and C-terminal flankingsequences as allele-noninteracting variables, with network dependencyfunctions g_(h)(⋅) and g_(w)(⋅), and the expit function ƒ(⋅). Thepresentation model for allele HLA-A*02:01 generates a presentationlikelihood that a given peptide will be presented on allele HLA-A*02:01,given the peptide sequence as an allele-interacting variable, and theN-terminal and C-terminal flanking sequences as allele-noninteractingvariables. The presentation model for allele HLA-B*07:02 generates apresentation likelihood that a given peptide will be presented on alleleHLA-B*07:02, given the peptide sequence as an allele-interactingvariable, and the N-terminal and C-terminal flanking sequences asallele-noninteracting variables.

As laid out in the following examples and with reference to FIGS.13A-13E, various models, such as the trained presentation models andcurrent state-of-the-art models for peptide binding prediction, areapplied to the test set of neoantigen candidates for each simulatedpatient to identify different treatment subsets for patients based onthe predictions. Patients that satisfy inclusion criteria are selectedfor vaccine treatment, and are associated with customized vaccines thatinclude epitopes in the treatment subsets of the patients. The size ofthe treatment subsets are varied according to different vaccinecapacities. No overlap is introduced between the training set used totrain the presentation model and the test set of simulated neoantigencandidates.

In the following examples, the proportion of selected patients having atleast a certain number of presented neoantigens among the epitopesincluded in the vaccines are analyzed. This statistic indicates theeffectiveness of the simulated vaccines to deliver potential neoantigensthat will elicit immune responses in patients. Specifically, a simulatedneoantigen in a test set is presented if the neoantigen is presented inthe mass spectrometry data set D2. A high proportion of patients withpresented neoantigens indicate potential for successful treatment vianeoantigen vaccines by inducing immune responses.

XI.A. Example 7A: Frequency Distribution of Mutation Burden for NSCLCCancer Patients

FIG. 13A illustrates a sample frequency distribution of mutation burdenin NSCLC patients. Mutation burden and mutations in different tumortypes, including NSCLC, can be found, for example, at the cancer genomeatlas (TCGA) (https://cancergenome.nih.gov). The x-axis represents thenumber of non-synonymous mutations in each patient, and the y-axisrepresents the proportion of sample patients that have the given numberof non-synonymous mutations. The sample frequency distribution in FIG.13A shows a range of 3-1786 mutations, in which 30% of the patients havefewer than 100 mutations. Although not shown in FIG. 13A, researchindicates that mutation burden is higher in smokers compared to that ofnon-smokers, and that mutation burden may be a strong indicator ofneoantigen load in patients.

As introduced at the beginning of Section XI above, each of a number ofsimulated patients are associated with a test set of neoantigencandidates. The test set for each patient is generated by sampling amutation burden m; from the frequency distribution shown in FIG. 13A foreach patient. For each mutation, a 21-mer peptide sequence from thehuman proteome is randomly selected to represent a simulated mutatedsequence. A test set of neoantigen candidate sequences are generated forpatient i by identifying each (8, 9, 10, 11)-mer peptide sequencespanning the mutation in the 21-mer. Each neoantigen candidate isassociated with a label indicating whether the neoantigen candidatesequence was present in the mass spectrometry D1 data set. For example,neoantigen candidate sequences present in data set D1 may be associatedwith a label “1,” while sequences not present in data set D1 may beassociated with a label “0.” As described in more detail below, FIGS.13B through 13E illustrate experimental results on patient selectionbased on presented neoantigens of the patients in the test set.

XI.B. Example 7B: Proportion of Selected Patients with NeoantigenPresentation Based on Mutation Burden Inclusion Criteria

FIG. 13B illustrates the number of presented neoantigens in simulatedvaccines for patients selected based on an inclusion criteria of whetherthe patients satisfy a minimum mutation burden. The proportion ofselected patients that have at least a certain number of presentedneoantigens in the corresponding test is identified.

In FIG. 13B, the x-axis indicates the proportion of patients excludedfrom vaccine treatment based on the minimum mutation burden, asindicated by the label “minimum # of mutations.” For example, a datapoint at 200 “minimum # of mutations” indicates that the patientselection module 324 selected only the subset of simulated patientshaving a mutation burden of at least 200 mutations. As another example,a data point at 300 “minimum # of mutations” indicates that the patientselection module 324 selected a lower proportion of simulated patientshaving at least 300 mutations. The y-axis indicates the proportion ofselected patients that are associated with at least a certain number ofpresented neoantigens in the test set without any vaccine capacity ν.Specifically, the top plot shows the proportion of selected patientsthat present at least 1 neoantigen, the middle plot shows the proportionof selected patients that present at least 2 neoantigens, and the bottomplot shows the proportion of selected patients that present at least 3neoantigens.

As indicated in FIG. 13B, the proportion of selected patients withpresented neoantigens increases significantly with higher mutationburden. This indicates that mutation burden as an inclusion criteria canbe effective in selecting patients for whom neoantigen vaccines are morelikely to induce successful immune responses.

XI.C. Example 7C: Comparison of Neoantigen Presentation for VaccinesIdentified by Presentation Models Vs. State-of-the-Art Models

FIG. 13C compares the number of presented neoantigens in simulatedvaccines between selected patients associated with vaccines includingtreatment subsets identified based on presentation models and selectedpatients associated with vaccines including treatment subsets identifiedthrough current state-of-the-art models. The left plot assumes limitedvaccine capacity v=10, and the right plot assumes limited vaccinecapacity v=20. The patients are selected based on utility scoresindicating expected number of presented neoantigens.

In FIG. 13C, the solid lines indicate patients associated with vaccinesincluding treatment subsets identified based on presentation models foralleles HLA-A*02:01 and HLA-B*07:02. The treatment subset for eachpatient is identified by applying each of the presentation models to thesequences in the test set, and identifying the v neoantigen candidatesthat have the highest presentation likelihoods. The dotted linesindicate patients associated with vaccines including treatment subsetsidentified based on current state-of-the-art models NETMHCpan for thesingle allele HLA-A*02:01. Implementation details for NETMHCpan isprovided in detail at http://www.cbs.dtu.dk/services/NetMHCpan. Thetreatment subset for each patient is identified by applying theNETMHCpan model to the sequences in the test set, and identifying the vneoantigen candidates that have the highest estimated bindingaffinities. The x-axis of both plots indicates the proportion ofpatients excluded from vaccine treatment based on expectation utilityscores indicating the expected number of presented neoantigens intreatment subsets identified based on presentation models. Theexpectation utility score is determined as described in reference toequation (25) in Section X. The y-axis indicates the proportion ofselected patients that present at least a certain number of neoantigens(1, 2, or 3 neoantigens) included in the vaccine.

As indicated in FIG. 13C, patients associated with vaccines includingtreatment subsets based on presentation models receive vaccinescontaining presented neoantigens at a significantly higher rate thanpatients associated with vaccines including treatment subsets based onstate-of-the-art models. For example, as shown in the right plot, 80% ofselected patients associated with vaccines based on presentation modelsreceive at least one presented neoantigen in the vaccine, compared toonly 40% of selected patients associated with vaccines based on currentstate-of-the-art models. The results indicate that presentation modelsas described herein are effective for selecting neoantigen candidatesfor vaccines that are likely to elicit immune responses for treatingtumors.

X.I.D. Example 7D: Effect of HLA Coverage on Neoantigen Presentation forVaccines Identified Through Presentation Models

FIG. 13D compares the number of presented neoantigens in simulatedvaccines between selected patients associated with vaccines includingtreatment subsets identified based on a single per-allele presentationmodel for HLA-A*02:01 and selected patients associated with vaccinesincluding treatment subsets identified based on both per-allelepresentation models for HLA-A*02:01 and HLA-B*07:02. The vaccinecapacity is set as v=20 epitopes. For each experiment, the patients areselected based on expectation utility scores determined based on thedifferent treatment subsets.

In FIG. 13D, the solid lines indicate patients associated with vaccinesincluding treatment subsets based on both presentation models for HLAalleles HLA-A*02:01 and HLA-B*07:02. The treatment subset for eachpatient is identified by applying each of the presentation models to thesequences in the test set, and identifying the v neoantigen candidatesthat have the highest presentation likelihoods. The dotted linesindicate patients associated with vaccines including treatment subsetsbased on a single presentation model for HLA allele HLA-A*02:01. Thetreatment subset for each patient is identified by applying thepresentation model for only the single HLA allele to the sequences inthe test set, and identifying the v neoantigen candidates that have thehighest presentation likelihoods. For solid line plots, the x-axisindicates the proportion of patients excluded from vaccine treatmentbased on expectation utility scores for treatment subsets identified byboth presentation models. For dotted line plots, the x-axis indicatesthe proportion of patients excluded from vaccine treatment based onexpectation utility scores for treatment subsets identified by thesingle presentation model. The y-axis indicates the proportion ofselected patients that present at least a certain number of neoantigens(1, 2, or 3 neoantigens).

As indicated in FIG. 13D, patients associated with vaccines includingtreatment subsets identified by presentation models for both HLA allelespresent neoantigens at a significantly higher rate than patientsassociated with vaccines including treatment subsets identified by asingle presentation model. The results indicate the importance ofestablishing presentation models with high HLA allele coverage.

XI.E. Example 7E: Comparison of Neoantigen Presentation for PatientsSelected by Mutation Burden Vs. Expected Number of Presented Neoantigens

FIG. 13E compares the number of presented neoantigens in simulatedvaccines between patients selected based on mutation burden and patientsselected by expectation utility score. The expectation utility scoresare determined based on treatment subsets identified by presentationmodels having a size of v=20 epitopes.

In FIG. 13E, the solid lines indicate patients selected based onexpectation utility score associated with vaccines including treatmentsubsets identified by presentation models. The treatment subset for eachpatient is identified by applying the presentation models to sequencesin the test set, and identifying the v=20 neoantigen candidates thathave the highest presentation likelihoods. The expectation utility scoreis determined based on the presentation likelihoods of the identifiedtreatment subset based on equation (25) in section X. The dotted linesindicate patients selected based on mutation burden associated withvaccines also including treatment subsets identified by presentationmodels. The x-axis indicates the proportion of patients excluded fromvaccine treatment based on expectation utility scores for solid lineplots, and proportion of patients excluded based on mutation burden fordotted line plots. The y-axis indicates the proportion of selectedpatients who receive a vaccine containing at least a certain number ofpresented neoantigens (1, 2, or 3 neoantigens).

As indicated in FIG. 13E, patients selected based on expectation utilityscores receive a vaccine containing presented neoantigens at a higherrate than patients selected based on mutation burden. However, patientsselected based on mutation burden receive a vaccine containing presentedneoantigens at a higher rate than unselected patients. Thus, mutationburden is an effective patient selection criteria for successfulneoantigen vaccine treatment, though expectation utility scores are moreeffective.

XII. Example 8: Evaluation of Mass Spectrometry-Trained Model onHeld-Out Mass Spectrometry Data

As HLA peptide presentation by tumor cells is a key requirement foranti-tumor immunity^(91,96,97), a large (N=74 patients) integrateddataset of human tumor and normal tissue samples with paired class I HLApeptide sequences, HLA types and transcriptome RNA-seq (Methods) wasgenerated with the aim of using these and publicly availabledata^(92,98,99) to train a novel deep learning model¹⁰⁰ to predictantigen presentation in human cancer. Samples were chosen among severaltumor types of interest for immunotherapy development and based ontissue availability. Mass spectrometry identified an average of 3,704peptides per sample at peptide-level FDR<0.1 (range 344-11,301). Thepeptides followed the characteristic class I HLA length distribution:lengths 8-15aa, with a modal length of 9 (56% of peptides). Consistentwith previous reports, a majority of peptides (median 79%) werepredicted to bind at least one patient HLA allele at the standard 500 nMaffinity threshold by MHCflurry⁹⁰, but with substantial variabilityacross samples (e.g., 33% of peptides in one sample had predictedaffinities >500 nM). The commonly used¹⁰¹ “strong binder” threshold of50 nM captured a median of only 42% of presented peptides. Transcriptomesequencing yielded an average of 131M unique reads per sample and 68% ofgenes were expressed at a level of at least 1 transcript per million(TPM) in at least one sample, highlighting the value of a large anddiverse sample set to observe expression of a maximal number of genes.Peptide presentation by the HLA was strongly correlated with mRNAexpression. Striking and reproducible gene-to-gene differences in therate of peptide presentation, beyond what could be explained bydifferences in RNA expression or sequence alone, were observed. Theobserved HLA types matched expectations for specimens from apredominantly European-ancestry group of patients.

Using these and publicly available HLA peptide dat^(92,98,99), a neuralnetwork (NN) model was trained to predict HLA antigen presentation. Tolearn allele-specific models from tumor mass spectrometry data whereeach peptide could have been presented by any one of six HLA alleles, anovel network architecture capable of jointly learning theallele-peptide mappings and allele-specific presentation motifs(Methods) was developed. For each patient, the positive-labeled datapoints were peptides detected via mass spectrometry, and thenegative-labeled data points were peptides from the reference proteome(SwissProt) that were not detected via mass spectrometry in that sample.The data was split into training, validation and testing sets (Methods).The training set consisted of 142,844 HLA presented peptides (FDR<˜0.02)from 101 samples (69 newly described in this study and 32 previouslypublished). The validation set (used for early stopping) consisted of18,004 presented peptides from the same 101 samples. Two massspectrometry datasets were used for testing: (1) A tumor sample test setconsisting of 571 presented peptides from 5 additional tumor samples (2lung, 2 colon, 1 ovarian) that were held out of the training data, and(2) a single-allele cell line test set consisting of 2,128 presentedpeptides from genomic location windows (blocks) adjacent to, butdistinct from, the locations of single-allele peptides included in thetraining data (see Methods for additional details on the train/testsplits).

The training data identified predictive models for 53 HLA alleles. Incontrast to prior work^(92,104), these models captured the dependence ofHLA presentation on each sequence position for peptides of multiplelengths. The model also correctly learned the critical dependencies ongene RNA expression and gene-specific presentation propensity, with themRNA abundance and learned per-gene propensity of presentation combiningindependently to yield up to a ˜60-fold difference in rate ofpresentation between the lowest-expressed, least presentation-prone andthe highest expressed, most presentation-prone genes. It was furtherobserved that the model predicted the measured stability of HLA/peptidecomplexes in IEDB⁸⁸ (p<1e-10 for 10 alleles), even after controlling forpredicted binding affinity (p<0.05 for 8/10 alleles tested).Collectively, these features form the basis for improved prediction ofimmunogenic HLA class I peptides.

Performance of this NN model as a predictor of HLA presentation on theheld-out mass spectrometry test sets was evaluated and compared to thelatest state-of-the-art binding affinity predictor MHCFlurry⁹⁰ (version1.2.0, Methods), a neural network tool trained on in vitro HLA bindingdata. Based on prior reports highlighting the importance of mRNA levelfor HLA presentation, increasing thresholds on gene expression asassayed by RNA-seq^(81,92,103) were incorporated.

FIGS. 14A-D compare predictive performance of the “Full MS Model,” the“Peptide MS Model,” and the MHCFlurry 1.2.0 binding affinity model withthree different gene expression thresholds. The “Full MS Model” and the“Peptide MS Model” are both neural network models trained on massspectrometry data as described above. However, the “Full MS Model” istrained and tested based on all features of a sample, while the “PeptideMS Model” is trained and tested based only on the HLA types and thepeptide sequences of a sample. Three distinct versions of the MHCFlurry1.2.0 binding affinity model are tested: a MHCFlurry 1.2.0 bindingaffinity model with a gene expression threshold of TPM >0, a MHCFlurry1.2.0 binding affinity model with a gene expression threshold of TPM >1,and a MHCFlurry 1.2.0 binding affinity model with a gene expressionthreshold of TPM >2. Because the “Peptide MS Model” and the MHCFlurry1.2.0 binding affinity model with a gene expression threshold of TPM >1are both trained and tested based only on the HLA types and the peptidesequences of a sample, and both have the same RNA expression threshold,comparing the performance of these two models directly quantifies thepredictive improvement attributable to differences in peptide motifslearned from mass spectrometry vs binding affinity training data.

Turning first to FIG. 14A, FIG. 14A compares the positive predictivevalues (PPV) at 40% recall of the “Full MS Model,” the “Peptide MSModel,” and the MHCFlurry 1.2.0 binding affinity model with the threedifferent gene expression thresholds of TPM >0, 1, and 2 when each modelis tested on a test set comprising five different test samples, eachtest sample comprising a held-out tumor sample with a 1:2500 ratio ofpresented to non-presented peptides (Methods). FIG. 14A also depicts theaverage PPV at 40% recall of the “Full MS Model,” the “Peptide MSModel,” and the MHCFlurry 1.2.0 binding affinity model with the threedifferent gene expression thresholds of TPM >0, 1, and 2, for the fivetest samples. As shown in FIG. 14A, the average PPV at 40% recall was0.54 for the “Full MS Model,” and 0.076, 0.072, and 0.061 for theMHCFlurry 1.2.0 binding affinity model with the gene expressionthresholds of TPM >2, 1, and 0, respectively. All comparisons betweenthe “Full MS Model” and the MHCFlurry 1.2.0 binding affinity model witha gene expression threshold of TPM >0 are statistically significant atp<1e-6.

Turning next to FIG. 14B, FIG. 14B compares PPV at 40% recall of the“Full MS Model,” the “Peptide MS Model,” and the MHCFlurry 1.2.0 bindingaffinity model with the three different gene expression thresholds ofTPM >0, 1, and 2, when each model is tested on a test set comprising 15different test samples, each test sample comprising held-out peptidesfrom a single-allele cell line test dataset with a 1:10.000 ratio ofpresented to non-presented peptides (Methods). FIG. 14B also depicts theaverage PPV at 40% recall of the “Full MS Model” the “Peptide MS Model,”and the MHCFlurry 1.2.0 binding affinity model with the three differentgene expression thresholds of TPM >0, 1, and 2, for the 15 test samples.As shown in FIG. 14B, the average PPV at 40% recall was 0.37 for the“Full MS Model,” and 0.094, 0.090, and 0.071 for the MHCFlurry 1.2.0binding affinity model with the gene expression thresholds of TPM >2, 1,and 0, respectively. All comparisons between the “Full MS Model” and theMHCFlurry 1.2.0 binding affinity model with a gene expression thresholdof TPM >0 are statistically significant at p<1e-6, except for the testsample comprising HLA-A*01:01, for which p=1.6e-4.

FIG. 16 compares the positive predictive values (PPV) at 40% recall ofthe “Full MS Model” and an “Anchor Residue Only MS Model,” when eachmodel is tested on the test set described above with regard to FIG. 14A(Methods). FIG. 16 also depicts the average PPV at 40% recall of the“Full MS Model” and the “Anchor Residue Only MS Model,” for the fivetest samples. Like the “Full MS Model,” the “Anchor Residue Only MSModel” is a neural network model trained on mass spectrometry data asdescribed above. However, rather than training and testing the “AnchorResidue Only MS Model” based on entire peptide sequences in a sample,the “Anchor Residue Only MS Model” is trained and tested based only onthe “anchor” residues (the first, second, and last residues) of thepeptide sequences of a sample. Thus the results depicted in FIG. 16quantify the relative importance of anchor and non-anchor residues tothe model's predictive performance. As shown in FIG. 16, the performanceof the “Anchor Residue Only MS Model” is substantially reduced comparedto the full MS model. The average PPV at 40% recall for the anchorresidue only MS model is 0.13, compared to 0.50 for the full MS model.Therefore, it can be deduced that training and testing of the model withnon-anchor residues of peptide sequences results in predictiveimprovement of the model.

FIG. 17A depicts full precision-recall curves for the “Full MS Model,”the “Peptide MS Model,” and the MHCFlurry 1.2.0 binding affinity modelwith the three different gene expression thresholds of TPM >0, 1, and 2when each model is tested on test sample 0 from FIG. 14A (Methods). Asshown in FIG. 17A, the “Full MS Model” and the “Peptide MS Model”achieve better performance than the MHCFlurry 1.2.0 binding affinitymodel with the three different gene expression thresholds of TPM >0, 1,and 2.

FIG. 17B compares PPV at 40% recall of the “Full MS Model,” the “PeptideMS Model,” and the MHCFlurry 1.2.0 binding affinity model with the threedifferent gene expression thresholds of TPM >0, 1, and 2, when eachmodel is tested on a test set comprising 15 different test samples, eachtest sample comprising held-out peptides from a single-allele cell linetest dataset with a 1:5,000 ratio of presented to non-presented peptides(Methods). FIG. 17B also depicts the average PPV at 40% recall of the“Full MS Model,” the “Peptide MS Model,” and the MHCFlurry 1.2.0 bindingaffinity model with the three different gene expression thresholds ofTPM >0, 1, and 2, for the 15 test samples. By comparing the results ofFIG. 14B (in which each test sample comprises held-out peptides from asingle-allele cell line test dataset with a 1:10,000 ratio of presentedto non-presented peptides) to the results of FIG. 17A (in which eachtest sample comprises held-out peptides from a single-allele cell linetest dataset with a 1:5.000 ratio of presented to non-presentedpeptides), it can be deduced that prevalence of peptide presentation isstrongly correlated with absolute PPV. In general, the lower theprevalence of the event to be predicted (e.g., presentation), the moredifficult it is to achieve high PPV prediction. Consequently, lowering(raising) the prevalence in the test data will lower (raise) theabsolute PPVs of all models. However, the relative differences betweenthe PPVs of different models are not affected by changes in test setprevalence in expectation.

FIGS. 17C-G depict full precision-recall curves for the “Full MS Model,”the “Peptide MS Model,” and the MHCFlurry 1.2.0 binding affinity modelwith the three different gene expression thresholds of TPM >0, 1, and 2when each model is tested on test samples 0-4 from FIG. 14A (Methods).

FIGS. 17H-V depict full precision-recall curves for the “Full MS Model,”the “Peptide MS Model,” and the MHCFlurry 1.2.0 binding affinity modelwith the three different gene expression thresholds of TPM >0, 1, and 2when each model is tested on the 15 different test samples of FIG. 14B,each test sample comprising held-out peptides from a single-allele cellline test dataset with a 1:10,000 ratio of presented to non-presentedpeptides (Methods).

FIG. 18 compares the positive predictive values (PPV) at 40% recall ofdifferent versions of the “MS Model and earlier approaches to modelingHLA presented peptides¹” in human tumors, when each model is tested onthe five different test samples of FIG. 14A (Methods). FIG. 18 alsodepicts the average PPV at 40% recall of the models for the five testsamples. The models tested in FIG. 18 include the “Full MS Model,” the“MS Model, No Flanking Sequence.” the “MS Model, No Flanking Sequence orPer-Gene Coefficient,” the “Peptide-Only MS Model, all Lengths TrainedJointly,” the “Peptide-Only MS Model, all Lengths Trained Separately,”the “Linear Peptide-Only MS Model,” the “MixMHCPred 1.1” model, and the“Binding Affinity” model. The “Full MS Model,” the “MS Model, NoFlanking Sequence,” the “MS Model, No Flanking Sequence or Per-GeneCoefficient,” the “Peptide-Only MS Model, all Lengths Trained Jointly,”the “Peptide-Only MS Model, all Lengths Trained Separately,” and the“Linear Peptide-Only MS Model” are all neural network models trained onmass spectrometry data as described above. However, each model istrained and tested using different features of a sample. The “MixMHCPred1.1” model and the “Binding Affinity” model are earlier approaches tomodeling HLA presented peptides¹⁰⁴.

Overall, the NN model achieved significantly improved prediction of HLApeptide presentation, with a PPV up to 9-fold higher than standardbinding affinity+gene expression on the tumor test set (FIG. 14A) and upto 5-fold higher on the single-allele dataset (FIG. 14B). The large PPVadvantage of the MS-based NN model persisted across various recallthresholds (FIG. 17A) and was statistically significant (p<10⁶ for alltumor and single-allele samples in FIGS. 14A and 14B except HLA-A*01:01,for which p=1.6e-4). The positive predictive value of standard bindingaffinity+gene expression for HLA peptide presentation reached as low as6%, in line with previous estimates^(87,93). Notably, however, this ˜6%PPV still represents a >100-fold enrichment over baseline prevalence,because only a small proportion of peptides are detected as presented(e.g., ˜1 in 2500 in the tumor MS test dataset).

By comparing a reduced model trained on mass spectrometry data that usesonly HLA type and peptide sequence as inputs (“Peptide MS Model”, FIGS.14A-B: see Methods) to the full MS model, it was determined that ˜30% ofthe gain in PPV over binding affinity prediction came from modelingpeptide-extrinsic features (RNA abundance, flanking sequence, per-genecoefficients) that can be captured with mass spectrometry but notbinding affinity assays (FIGS. 14A-B; also see FIGS. 17A and 18). Theother ˜70% of the gain came from improved modeling of peptide sequence(FIGS. 14A-B). It was not just the nature of the training dataset (HLApresented peptides), but the overall model architecture that contributedto the improved performance, as it also outperformed earlier approachesto modeling HLA presented peptides¹⁰⁴ in human tumors (FIG. 18). The newmodel architecture enabled learning of allele-specific models via anend-to-end training process that does not require ex ante assignment ofpeptides to purported presenting alleles using binding affinitypredictions or hard-clustering approaches¹⁰⁴⁻¹⁰⁶. Importantly, it alsoavoided imposing accuracy-reducing restrictions on the allele-specificsub-models as a prerequisite to deconvolution, such as linearity, orseparate consideration of each peptide length¹⁰⁴, the full modeloutperforms several simplified models and previously publishedapproaches that impose these restrictions (FIG. 18).

FIG. 18 illustrates the performance of several simplified models on theMS test sets. The relative importance of the modeling improvementsincorporated in the full model are quantified by removing modelingimprovements one-at-a-time and testing predictive performance on the MStest set. In addition, a comparison of the presentation model disclosedherein to a recently published approach to modeling eluted peptides frommass spectrometry (MixMHCPred) was performed. Only 9 and 10 mers wereused in the comparison because MixMHCPred does not currently modelpeptides of lengths other than 9 and 10. The models are (from left toright): “Full MS Model”: the full NN model described in the Methods: “MSModel, No Flanking Sequence”: identical to the full NN model, exceptwith the flanking sequence feature removed; “MS Model, No FlankingSequence or Per-Gene Coefficients”; identical to the full NN model,except with the flanking sequence and per-gene coefficient featuresremoved; “Peptide-Only MS Model, all Lengths Trained Jointly”: identicalto the full NN model, except the only features used are peptide sequenceand HLA type; “Peptide-Only MS Model, Each Length Trained Separately”:for this model, the model structure was the same as the peptide-only MSmodel, except separate models for 9 and 10 mers were trained; “LinearPeptide-Only MS Model (with Ensembling)”: identical to the peptide-onlyMS model with each peptide length trained separately; except instead ofmodeling peptide sequence using neural networks, an ensemble of linearmodels trained using the same optimization procedure used for the fullmodel and described in the Methods was used: “MixMHCPred 1.1” isMixMHCPred with default settings; “Binding affinity” is MHCflurry 1.2.0,as in the main text. The last 5 models (“Peptide-Only MS Model, allLengths Trained Jointly” through “Binding Affinity”) have the sameinputs: peptide sequence and HLA types, only. In particular, none of thelast 5 models uses RNA abundance to make predictions. The bestperforming peptide-only model (“Peptide-Only MS Model, all LengthsTrained Jointly) achieves an average PPV of 0.41 at 40% recall, whilethe worst-performing peptide-only model trained on the mass spectrometrydata (“Linear Peptide-Only MS Model (with Ensembling)” achieves anaverage PPV of only 28% (only slightly higher than to the average PPV ofMixMHCpred at 18%), highlighting the value of improved NN modeling ofpeptide sequences. Note that MixMHCpred is trained on different datathan the linear peptide-only MS model, but has many of the same modelingcharacteristics (e.g., it is a linear model, where the models for eachpeptide length are trained separately).

XIII. Example 9: Model Evaluation of Retrospective Neoantigen T-CellData

We then evaluated whether this accurate prediction of HLA peptidepresentation translated into the ability to identify human tumor CD8T-cell epitopes (i.e., immunotherapy targets). An appropriate testdataset for this evaluation includes peptides that are both recognizedby T-cells and presented by the HLA on the tumor cell surface. Inaddition, formal performance assessment requires not onlypositive-labeled (i.e., T-cell recognized) peptides, but also asufficient number of negative-labeled (i.e., tested but not recognized)peptides. Mass spectrometry datasets address tumor presentation but notT-cell recognition; oppositely, priming or T-cell assayspost-vaccination address the presence of T-cell precursors and T-cellrecognition but not tumor presentation. For example, a strongHLA-binding peptide whose source gene is expressed at low level in thetumor could give rise to a strong CD8 T-cell response post-immunizationthat would not be therapeutically useful because the peptide is notpresented by the tumor.

To obtain an appropriate dataset, published CD8 T-cell epitopes werecollected from 4 recent studies that met the required criteria: studyA⁹⁶ examined TIL in 9 patients with gastrointestinal tumors and reportedT-cell recognition of 12/1.053 somatic SNV mutations tested by IFN-gammaELISPOT using the tandem minigene (TMG) method in autologous dendriticcells (DCs). Study B¹⁰⁷ also used TMGs and reported T-cell recognitionof 6/574 SNVs by CD8+PD-1+ circulating lymphocytes from 4 melanomapatients. Study C⁹⁷ assessed TIL from 3 melanoma patients using pulsedpeptide stimulation and found responses to 5/381 tested SNV mutations.Study D¹⁰⁸ assessed TIL from one breast cancer patient using acombination of TMG assays and pulsing with minimal epitope peptides andreported recognition of 2/62 SNVs. The combined dataset consisted of2,009 assayed SNVs from 17 patients, including 26 neoantigens withpre-existing T-cell responses. Importantly, because the datasetcomprises largely neoantigen recognition by tumor-infiltratinglymphocytes, successful prediction implies the ability to identify notjust neoantigens that are able to prime T-cells as in previousliterature^(81,82,97), but—more stringently—neoantigens presented toT-cells by tumors.

To simulate the selection of antigens for personalized immunotherapy,somatic mutations were ranked in order of probability of presentationusing the “Full MS Model,” the “Peptide MS Model,” and the MHCFlurry1.2.0 binding affinity model with the three different gene expressionthresholds of TPM >0, 1, and 2. As antigen-specific immunotherapies aretechnically limited in the number of specificities targeted (e.g.,current personalized vaccines encode ˜10-20 somatic mutations⁸⁰⁻⁸²),predictive methods were compared by counting the number of pre-existingT-cell responses in the top 5, 10, or 20-ranked somatic mutations foreach patient with at least one pre-existing T-cell repsonse. Theseresults are depicted in FIG. 14C. Specifically, FIG. 14C compares theproportion of somatic mutations recognized by T-cells (e.g.,pre-existing T-cell responses) for the top 5, 10, and 20-ranked somaticmutations identified by the “Full MS Model.” the “Peptide MS Model,” andthe MHCFlurry 1.2.0 binding affinity model with the three different geneexpression thresholds of TPM >0, 1, and 2 for a test set comprising 12different test samples, each test sample taken from a patient with atleast one pre-existing T-cell response. All comparisons between the“Full MS Model” and the MHCFlurry 1.2.0 binding affinity model with agene expression threshold of TPM >0 are statistically significant atp<0.005, except for the somatic mutations ranked in the top 5, for whichp=0.056.

As expected, binding affinity prediction included only a minority ofpre-existing T-cell responses among the prioritized mutations, forinstance 9 of the total 26 (35%) among the top 20 ranked mutations atTPM>0 (Supplementary Table 1). In contrast, the majority (19/26, 73%) ofpre-existing T-cell responses were ranked in the top 20 by the full MSmodel, and the advantage persisted across different rank and geneexpression thresholds (FIG. 14C, Supplementary Table 1). At the patientlevel, the full MS model averaged 1.54 pre-existing neoantigen T-cellresponses in the top 20 predicted mutations for the 13 patients with atleast one pre-existing T-cell response, compared to only 0.69 forbinding affinity at TPM>0 (p=1.4e-4).

We then evaluated mutations at the level of minimal neoepitopes (i.e.,which 8-11-mer overlapping the mutation was recognized), as may beuseful to identify T-cells/TCRs for cell therapy. In other words,minimal neoepitopes were ranked in order of probability of presentationusing the “Full MS Model.” the “Peptide MS Model,” and the MHCFlurry1.2.0 binding affinity model with the three different gene expressionthresholds of TPM >0, 1, and 2. As mentioned above, as antigen-specificimmunotherapies are technically limited in the number of specificitiestargeted, predictive methods were compared by counting the number ofpre-existing T-cell responses in the top 5, 10, or 20-ranked minimalneoepitopes for each patient with at least one pre-existing T-cellresponse. Positively-labeled epitopes were those confirmed to beimmunogenic minimal epitopes via peptide-based (instead of, or inaddition to, TMG-based assays), and negative examples were all epitopesnot recognized in peptide-based assays and all mutation-spanningepitopes contained in non-recognized minigenes. The results are decpitedin FIG. 14D.

Specifically, FIG. 14D compares the proportion of minimal neoepitopesrecognized by T-cells (e.g., pre-existing T-cell responses) for the top5, 10, and 20-ranked minimal neoepitopes identified by the “Full MSModel,” the “Peptide MS Model,” and the MHCFlurry 1.2.0 binding affinitymodel with the three different gene expression thresholds of TPM >0, 1,and 2 for a test set comprising 12 different test samples, each testsample taken from a patient with at least one pre-existing T-cellresponse. All comparisons between the “Full MS Model” and the MHCFlurry1.2.0 binding affinity model with a gene expression threshold of TPM >0are statistically significant at p<0.05, except for the minimalneoepitopes ranked in the top 5, for which p=0.082. In all panels, errorbars represent 90% confidence intervals.

As shown in FIG. 14D, the advantage of the NN model over bindingaffinity at TPM>0 was even more pronounced than in FIG. 14C: at least4-fold more neoepitopes were included in the top ranked minimalepitopes. Notably, this comparison is biased in favor of bindingaffinity prediction, as only peptides with strong binding affinitieswere tested individually in studies A. B and D. It is possible thatthere were T-cell recognized peptides with weak predicted HLA bindingaffinities that were never assayed in these studies, but would have beenselected by the model. Such peptides were observed in this study and arediscussed in detail below with regard to FIG. 15A and SupplementaryTable 3.

Despite known limitations of mass spectrometry in detecting peptidescontaining cysteine^(92,104), the NN model nevertheless outperformedbinding affinity prediction on cysteine-containing T-cell-recognizedepitopes, ranking 3 out of 7 cysteine-containing epitopes (43%) of inthe top 5, compared to 1 out of 7 in the top 5 for binding affinity withTPM >0 gene expression threshold. As with the mass spectrometry testset, the additional features that can be modeled given mass spectrometrytraining data (RNA, flanking sequence, per-gene coefficients) made asubstantial contribution to the increase in predictive performance;however, as in the mass spectrometry test data, the predictiveperformance of the peptide-only MS model was substantially improvedrelative to binding affinity prediction, indicating that a majority ofthe improvement came from improved modeling of peptide sequence (FIGS.14C-D, compare light blue bar to green bars).

Notably, this improvement was observed despite potential enrichment oftest set neoepitope false negatives (i.e., neoepitopes presented bytumors that are capable of being recognized by T-cells, but where aT-cell response was not detected) due to limitations of current TILassays. These limitations may include: (a) an immunosuppressive tumormicroenvironment and inefficient T-cell priming, (b) neoepitope-reactiveT-cell exhaustion, (c) TIL production of cytokines other than IFNg, and(d) heterogeneity in the tumor fractions used. It is therefore possiblethat the absolute predictive performance in terms of number ofimmunogenic peptides in the top 5-20 described here is pessimisticrelative to other contexts, e.g., administration of a potent neoantigencancer vaccine.

XIII.A. Data

We obtained mutation calls, HLA types and T-cell recognition data fromthe supplementary information of Gros et al⁸⁴, Tran et al¹⁴⁰, Stronen etal¹⁴¹ and Zacharakis et al. Patient-specific RNA-seq data wereunavailable. Reasoning that tumor RNA expression is correlated acrossdifferent patients with the same tumor type, RNA-seq data fromtumor-type-matched patients from TCGA was substituted, which was usedboth in the neural network predictions and for RNA expression filteringat TPM>1 before binding affinity prediction. The addition of tumor-typematched RNA-seq data improved predictive performance (FIGS. 14C-D).

For the mutation-level analysis (FIG. 14C), the positive-labeleddatapoints for Gros et al. Tran et al and Zacharakis et al weremutations recognized by patient T-cells in both the TMG assay or theminimal epitope peptide-pulsing assays. The negative-labeled datapointswere all other mutations tested in TMG assays. For Stronen et al, thepositive labeled mutations were mutations spanned by at least onerecognized peptide, and the negative datapoints were all mutationstested but not recognized in the tetramer assays. For the Gros, Tran andZacharakis data, mutations were ranked either by summing probabilitiesof presentation or taking the minimum binding affinity across allmutation-spanning peptides, as the mutated-25 mer TMG assay tests theT-cell recognition of all peptides spanning the mutation. For theStronen data, mutations were ranked either by summing probabilities ofpresentation or taking the minimum binding affinity across allmutation-spanning peptides tested in the tetramer assays. The full listof mutations and features is available in Supplementary Table 1.

For the epitope-level analysis, the positive-labeled datapoints were allminimal epitopes recognized by patient T-cells in peptide-pulsing ortetramer assays, and the negative datapoints were all minimal epitopesnot recognized by T-cells in peptide-pulsing or tetramer assays and allmutation-spanning peptides from tested TMGs that were not recognized bypatient T-cells. In the case of Gros et al, Tran et al and Zacharakis etal minimal epitope peptides spanning mutations recognized in the TMGanalysis that were not tested via peptide-pulsing assays were removedfrom the analysis, as the T-cell recognition status of these peptideswas not determined experimentally.

XIV. Example 10: Identification of NeoantiQen-Reactive T-Cells in CancerPatients

This example demonstrates that improved prediction can enable neoantigenidentification from routine patient samples. To do so, archival FFPEtumor biopsies and 5-30 ml of peripheral blood were analyzed from 9patients with metastatic NSCLC undergoing anti-PD(L)1 therapy(Supplementary Table 2: Patient demographics and treatment informationfor N=9 patients studied in FIGS. 15A-C. Key fields include tumor stageand subtype, anti-PD1 therapy received, and summary of NGS results).Tumor whole exome sequencing, tumor transcriptome sequencing, andmatched normal exome sequencing resulted in an average of 198 somaticmutations per patient (SNVs and short indel), of which an average of 118were expressed (Methods, Supplementary Table 2). The full MS model wasapplied to prioritize 20 neoepitopes per patient for testing againstpre-existing anti-tumor T-cell responses. To focus the analysis onlikely CD8 responses, the prioritized peptides were synthesized as 8-11mer minimal epitopes (Methods), and then peripheral blood mononuclearcells (PBMCs) were cultured with the synthesized peptides in short invitro stimulation (IVS) cultures to expand neoantigen-reactive T-cells(Supplementary Table 3). After two weeks the presence ofantigen-specific T-cells was assessed using IFN-gamma ELISpot againstthe prioritized neoepitopes. In seven patients for whom sufficient PBMCswere available, separate experiments were also performed to fully orpartially deconvolve the specific antigens recognized. The results aredepicted in FIGS. 15A-C and 19A-22.

FIG. 15A depicts detection of T-cell responses to patient-specificneoantigen peptide pools for nine patients. For each patient, predictedneoantigens were combined into 2 pools of 10 peptides each according tomodel ranking and any sequence homologies (homologous peptides wereseparated into different pools). Then, for each patient, the in vitroexpanded PBMCs for the patient were stimulated with the 2patient-specific neoantigen peptide pools in IFN-gamma ELISpot. Data inFIG. 15A are presented as spot forming units (SFU) per 105 plated cellswith background (corresponding DMSO negative controls) subtracted.Background measurements (DMSO negative controls) are shown in FIG. 22.Responses of single wells (patients 1-038-001, CU02, CU03 and 1-050-001)or replicates with mean and standard deviation (all other patients)against cognate peptide pools #1 and #2 are shown for patients1-038-001, 1-050-001, 1-001-002, CU04, 1-024-001, 1-024-002 and CU05.For patients CU02 and CU03, cell numbers allowed testing againstspecific peptide pool #1 only. Samples with values >2-fold increaseabove background were considered positive and are designated with a star(responsive donors include patients 1-038-001, CU04, 1-024-001,1-024-002, and CU02). Unresponsive donors include patients 1-050-001,1-001-002, CU05, and CU03. FIG. 15 C depicts photographs of ELISpotwells with in vitro expanded PBMCs from patient CU04, stimulated inIFN-gamma ELISpot with DMSO negative control, PHA positive control,CU04-specific neoantigen peptide pool #1, CU04-specific peptide 1,CU04-specific peptide 6, and CU04-specific peptide 8.

FIGS. 19A-B depict results from control experiments with patientneoantigens in HLA-matched healthy donors. The results of theseexperiments verify that in vitro culture conditions expanded onlypre-existing in vivo primed memory T-cells, rather than enabling de novopriming in vitro.

FIG. 20 depicts detection of T-cell responses to PHA positive controlfor each donor and each in vitro expansion depicted in FIG. 15A. Foreach donor and each in vitro expansion in FIG. 15A, the in vitroexpanded patient PBMCs were stimulated with PHA for maximal T-cellactivation. Data in FIG. 20 are presented as spot forming units (SFU)per 10⁵ plated cells with background (corresponding DMSO negativecontrols) subtracted. Responses of single wells or biological replicatesare shown for patients 1-038-001, 1-050-001, 1-001-002, CU04, 1-024-001,1-024-002, CU05 and CU03. Testing with PHA was not conducted for patientCU02. Cells from patient CU02 were included into analyses, as a positiveresponse against peptide pool #1 (FIG. 15A) indicated viable andfunctional T-cells. As shown in FIG. 15A, donors that were responsive topeptide pools include patients 1-038-001, CU04, 1-024-001, and1-024-002. As also shown in FIG. 15A, donors that were unresponsive topeptide pools include patients 1-050-001, 1-001-002, CU05, and CU03.

FIG. 21A depicts detection of T-cell responses to each individualpatient-specific neoantigen peptide in pool #2 for patient CU04. FIG.21A also depicts detection of T-cell responses to PHA positive controlfor patient CU04. (This is positive control data is also shown in FIG.20.) For patient CU04, the in vitro expanded PBMCs for the patient werestimulated in IFN-gamma ELISpot with patient-specific individualneoantigen peptides from pool #2 for patient CU04. The in vitro expandedPBMCs for the patient were also stimulated in IFN-gamma ELISpot with PHAas a positive control. Data are presented as spot forming units (SFU)per 10⁵ plated cells with background (corresponding DMSO negativecontrols) subtracted.

FIG. 21B depicts detection of T-cell responses to individualpatient-specific neoantigen peptides for each of three visits of patientCU04 and for each of two visits of patient 1-024-002, each visitoccurring at a different time point. For both patients, the in vitroexpanded PBMCs for the patient were stimulated in IFN-gamma ELISpot withpatient-specific individual neoantigen peptides. For each patient, datafor each visit are presented as cumulative (added) spot forming units(SFU) per 105 plated cells with background (corresponding DMSO controls)subtracted. Data for patient CU04 are shown as background subtractedcumulative SFU from 3 visits. For patient CU04, background subtractedSFU are shown for the initial visit (T0) and subsequent visits 2 months(T0+2 months) and 14 months (T0+14 months) after the initial visit (T0).Data for patient 1-024-002 are shown as background subtracted cumulativeSFU from 2 visits. For patient 1-024-002, background subtracted SFU areshown for the initial visit (T0) and a subsequent visit 1 month (T0+1month) after the initial visit (T0). Samples with values >2-foldincrease above background were considered positive and are designatedwith a star.

FIG. 21C depicts detection of T-cell responses to individualpatient-specific neoantigen peptides and to patient-specific neoantigenpeptide pools for each of two visits of patient CU04 and for each of twovisits of patient 1-024-002, each visit occurring at a different timepoint. For both patients, the in vitro expanded PBMCs for the patientwere stimulated in IFN-gamma ELISpot with patient-specific individualneoantigen peptides as well as with patient-specific neoantigen peptidepools. Specifically, for patient CU04, the in vitro expanded PBMCs forpatient CU04 were stimulated in IFN-gamma ELISpot with CU04-specificindividual neoantigen peptides 6 and 8 as well as with CU04-specificneoantigen peptide pools, and for patient 1-024-002, the in vitroexpanded PBMCs for patient 1-024-002 were stimulated in IFN-gammaELISpot with 1-024-002-specific individual neoantigen peptide 16 as wellas with 1-024-002-specific neoantigen peptide pools. The data of FIG.21C are presented as spot forming units (SFU) per 105 plated cells withbackground (corresponding DMSO controls) subtracted for each technicalreplicate with mean and range. Data for patient CU04 are shown asbackground subtracted SFU from 2 visits. For patient CU04, backgroundsubtracted SFU are shown for the initial visit (T0: technicaltriplicates) and a subsequent visit at 2 months (T0+2 months; technicaltriplicates) after the initial visit (T0). Data for patient 1-024-002are shown as background subtracted SFU from 2 visits. For patient1-024-002, background subtracted SFU are shown for the initial visit(T0; technical triplicates) and a subsequent visit 1 month (T0 +1 month;technical duplicates, except for the sample stimulated with patient1-024-002-specific neoantigen peptide pools) after the initial visit(T0).

FIG. 22 depicts detection of T-cell responses to the twopatient-specific neoantigen peptide pools and to DMSO negative controlsfor the patients of FIG. 15A. For each patient, the in vitro expandedPBMCs for the patient were stimulated with the two patient-specificneoantigen peptide pools in IFN-gamma ELISpot. For each donor and eachin vitro expansion, the in vitro expanded patient PBMCs were alsostimulated in IFN-gamma ELISpot with DMSO as a negative control. Data inFIG. 22 are presented as spot forming units (SFU) per 105 plated cellswith background (corresponding DMSO negative controls) included forpatient-specific neoantigen peptide pools and corresponding DMSOcontrols. Responses of single wells (1-038-001, CU2, CU03 and 1-050-001)or average with standard deviation of biological duplicates (all othersamples) against cognate peptide pools #1 and #2 are shown for patients1-038-001, 1-050-001, 1-001-002, CU04, 1-024-001, 1-024-002 and CU05.For patients CU02 and CU03, cell numbers allowed testing againstspecific peptide pool #1 only. Samples with values >2-fold increaseabove background were considered positive and are designated with a star(responsive donors include patients 1-038-001, CU04, 1-024-001,1-024-002, and CU02). Unresponsive donors include patients 1-050-001,1-001-002, CU05, and CU03.

As discussed briefly above with regard to FIGS. 19A-B, to verify thatthe in vitro culture conditions expanded only pre-existing in vivoprimed memory T-cells, rather than enabling de novo priming in vitro, aseries of control experiments were performed with neoantigens inHLA-matched healthy donors. The results of these experiments aredepicted in FIGS. 19A-B and in Supplementary Table 5. The results ofthese experiments confirmed the absence of de novo priming and absenceof a detectable neoantigen-specific T-cell response in healthy donorsusing IVS culture technique.

By contrast, pre-existing neoantigen-reactive T-cells were identified inthe majority (5/9, 56%) of patients tested with patient-specific peptidepools (FIGS. 15A and 20-22) using IFN-gamma ELiSpot. Of the 7 patientsfor whom cell numbers permitted complete or partial testing ofindividual neoantigen cognate peptides, 4 patients responded to at leastone of the tested neoantigen peptides, and all of these patients had acorresponding pool response (FIG. 15B). The remaining 3 patients testedwith individual neoantigens (patients 1-001-002, 1-050-001 and CU05) hadno detectable responses against single peptides (data not shown),confirming the lack of response seen for these patients againstneoantigen pools (FIG. 15A). Among the 4 responsive patients, samplesfrom a single visit were available for 2 patients with a response(patients 1-024-001 and 1-038-001), while samples from multiple visitswere available for the other 2 patients with a response (CU04 and1-024-002). For the 2 patients with samples from multiple visits, thecumulative (added) spot forming units (SFU) from 3 visits (patient CU04)or 2 visits (patient 1-024-002) are shown in FIG. 15B and broken down byvisit in FIG. 21B. Additional PBMC samples from the same visits werealso available for patients 1-024-002 and CU04, and repeat IVS cultureand ELISpot confirmed responses to patient-specific neoantigens (FIG.21C).

Overall, among patients for whom at least one T-cell recognizedneoepitope was identified as shown by a response to a pool of 10peptides in FIG. 15A, the number of recognized neoepitopes averaged atleast 2 per patient (minimum of 10 epitopes identified in 5 patients,counting a recognized pool that could not be deconvolved as 1 recognizedpeptide). In addition to testing for IFN-gamma response by ELISpot,culture supernatants were also tested for granzyme B by ELISA and forTNF-alpha, IL-2 and IL-5 by MSD cytokine multiplex assay. Cells from 4of the 5 patients with positive ELISpots secreted 3 or more analytes,including granzyme B (Supplementary Table 4), indicatingpolyfunctionality of neoantigen-specific T-cells. Importantly, becausethe combined prediction and IVS method did not rely on a limited set ofavailable MHC multimers, responses were tested broadly acrossrestricting HLA alleles. Furthermore, this approach directly identifiesthe minimal epitope, in contrast to tandem minigene screening, whichidentifies recognized mutations, and requires a separate deconvolutionstep to identify minimal epitopes. Overall, the neoantigenidentification yield was comparable to previous best methods⁹⁶ testingTIL against all mutations with apheresis samples, while screening only20 synthetic peptides with a routine 5-30 mL of whole blood.

XIV.A. Peptides

Custom-made, recombinant lyophilized peptides were purchased from JPTPeptide Technologies (Berlin, Germany) or Genscript (Piscataway, N.J.,USA) and reconstituted at 10-50 mM in sterile DMSO (VWR International,Pittsburgh, Pa., USA), aliquoted and stored at −80° C.

XIV.B. Human Peripheral Blood Mononuclear Cells (PBMCs)

Cryopreserved HLA-typed PBMCs from healthy donors (confirmed HIV, HCVand HBV seronegative) were purchased from Precision for Medicine(Gladstone, N.J., USA) or Cellular Technology, Ltd. (Cleveland, Ohio,USA) and stored in liquid nitrogen until use. Fresh blood samples werepurchased from Research Blood Components (Boston, Mass., USA), leukopaksfrom AllCells (Boston, Mass., USA) and PBMCs were isolated byFicoll-Paque density gradient (GE Healthcare Bio, Marlborough, Mass.,USA) prior to cryopreservation. Patient PBMCs were processed at localclinical processing centers according to local clinical standardoperating procedures (SOPs) and IRB approved protocols. Approving IRBswere Quorum Review IRB, Comitato Etico Interaziendale A.O.U. San LuigiGonzaga di Orbassano, and Comité Ético de la Investigación del GrupoHospitalario Quirón en Barcelona.

Briefly, PBMCs were isolated through density gradient centrifugation,washed, counted, and cryopreserved in CryoStor CS10 (STEMCELLTechnologies, Vancouver, BC, V6A 1B6, Canada) at 5×10⁶ cells/ml.Cryopreserved cells were shipped in cryoports and transferred to storagein LN₂ upon arrival. Patient demographics are listed in SupplementaryTable 2. Cryopreserved cells were thawed and washed twice in OpTmizerT-cell Expansion Basal Medium (Gibco, Gaithersburg, Md., USA) withBenzonase (EMD Millipore, Billerica, Mass., USA) and once withoutBenzonase. Cell counts and viability were assessed using the GuavaViaCount reagents and module on the Guava easyCyte HT cytometer (EMDMillipore). Cells were subsequently re-suspended at concentrations andin media appropriate for proceeding assays (see next sections).

XIV.C. In Vitro Stimulation (IVS) Cultures

Pre-existing T-cells from healthy donor or patient samples were expandedin the presence of cognate peptides and IL-2 in a similar approach tothat applied by Ott et al.⁸¹ Briefly, thawed PBMCs were rested overnightand stimulated in the presence of peptide pools (10 μM per peptide, 10peptides per pool) in ImmunoCul™-XF T-cell Expansion Medium (STEMCELLTechnologies) with 10 IU/ml rhIL-2 (R&D Systems Inc., Minneapolis,Minn.) for 14 days in 24-well tissue culture plates. Cells were seededat 2×10⁶ cells/well and fed every 2-3 days by replacing 2/3 of theculture media. One patient sample showed a deviation from the protocoland should be considered as a potential false negative: Patient CU03 didnot yield sufficient numbers of cells post thawing and cells were seededat 2×10⁵ cells per peptide pool (10-fold fewer than per protocol).

XIV.D. IFNfγ Enzyme Linked Immunospot (ELISpot) Assay

Detection of IFNγ-producing T-cells was performed by ELISpot assay¹⁴².Briefly, PBMCs (ex vivo or post in vitro expansion) were harvested,washed in serum free RPMI (VWR International) and cultured in thepresence of controls or cognate peptides in OpTmizer T-cell ExpansionBasal Medium (ex vivo) or in ImmunoCult™-XF T-cell Expansion Medium(expanded cultures) in ELISpot Multiscreen plates (EMD Millipore) coatedwith anti-human IFNγ capture antibody (Mabtech, Cincinatti, Ohio, USA).Following 18 h incubation in a 5% CO₂, 37° C., humidified incubator,cells were removed from the plate and membrane-bound IFNγ was detectedusing anti-human IFNγ detection antibody (Mabtech), Vectastain Avidinperoxidase complex (Vector Labs, Burlingame, Calif., USA) and AECSubstrate (BD Biosciences, San Jose, Calif., USA). ELISpot plates wereallowed to dry, stored protected from light and sent to ZellnetConsulting. Inc., Fort Lee, N.J., USA) for standardized evaluation¹⁴³.Data are presented as spot forming units (SFU) per plated number ofcells.

XIV.E. Granzyme B ELISA and MSD Multiplex Assay

Detection of secreted IL-2, IL-5 and TNF-alpha in ELISpot supernatantswas performed using using a 3-plex assay MSD U-PLEX Biomarker assay(catalog number K15067L-2). Assays were performed according to themanufacturer's instructions. Analyte concentrations (pg/ml) werecalculated using serial dilutions of known standards for each cytokine.For graphical data representation, values below the minimum range of thestandard curve were represented equals zero. Detection of Granzyme B inELISpot supernatants was performed using the Granzyme B DuoSet® ELISA (R& D Systems. Minneapolis, Minn.) according to the manufacturer'sinstructions. Briefly. ELISpot supernatants were diluted 1:4 in samplediluent and run alongside serial dilutions of Granzyme B standards tocalculate concentrations (pg/ml). For graphical data representation,values below the minimum range of the standard curve were representedequals zero.

XIV.F. Negative Control Experiments for IVS Assay—Neoantigens from TumorCell Lines Tested in Healthy Donors

FIG. 19A illustrates negative control experiments for IVS assay forneoantigens from tumor cell lines tested in healthy donors. Healthydonor PBMCs were stimulated in IVS culture with peptide pools containingpositive control peptides (previous exposure to infectious diseases),HLA-matched neoantigens originating from tumor cell lines (unexposed),and peptides derived from pathogens for which the donors wereseronegative. Expanded cells were subsequently analyzed by IFNγ ELISpot(10⁵ cells/well) following stimulation with DMSO (negative controls,black circles), PHA and common infectious diseases peptides (positivecontrols, red circles), neoantigens (unexposed, light blue circles), orHIV and HCV peptides (donors were confirmed to be seronegative, navyblue, A and B). Data are shown as spot forming units (SFU) per 105seeded cells. Biological replicates with mean and SEM are shown. Noresponses were observed to neoantigens or to peptides deriving frompathogens to which the donors have not been exposed (seronegative).

XIV.G. Negative Control Experiments for IVS Assay—Neoantigens fromPatients Tested in Healthy Donors

FIG. 19A illustrates negative control experiments for IVS assay forneoantigens from patients tested for reactivity in healthy donors.Assessment of T-cell responses in healthy donors to HLA-matchedneoantigen peptide pools. Left panel: Healthy donor PBMCs werestimulated with controls (DMSO, CEF and PHA) or HLA-matchedpatient-derived neoantigen peptides in ex vivo IFN-gamma ELISpot. Dataare presented as spot forming units (SFU) per 2×10⁵ plated cells fortriplicate wells. Right panel: Healthy donor PBMCs post IVS culture,expanded in the presence of either neoantigen pool or CEF pool werestimulated in IFN-gamma ELISpot either with controls (DMSO. CEF and PHA)or HLA-matched patient-derived neoantigen peptide pool. Data arepresented as SFU per 1×10⁵ plated cells for triplicate wells. Noresponses to neoantigens in healthy donors are seen.

XIV.H. Supplementary Table 3: Peptides Tested for T-Cell Recognition inNSCLC Patients

Details on neoantigen peptides tested for the N=9 patients studied inFIGS. 15A-C (Identification of Neoantigen-Reactive T-cells from NSCLCPatients). Key fields include source mutation, peptide sequence, andpool and individual peptide responses observed. The“most_probable_restriction” column indicates which allele the modelpredicted was most likely to present each peptide. The ranks of thesepeptides among all mutated peptides for each patient as computed withbinding affinity prediction (Methods) are also included.

There were four peptides highly ranked by the full MS model andrecognized by CD8 T-cells that had low predicted binding affinities orwere ranked low by binding affinity prediction.

For three of these peptides, this is caused by differences in HLAcoverage between the model and MHCflurry 1.2.0. Peptide YEHEDVKEA (SEQID NO: 20) is predicted to be presented by HLA-B*49:01, which is notcovered by MHCflurry 1.2.0. Similarly, peptides SSAAAPFPL (SEQ ID NO:21) and FVSTSDIKSM (SEQ ID NO: 22) are predicted to be presented byHLA-C*03:04, which is also not covered by MHCflurry 1.2.0. The onlineNetMHCpan 4.0 (BA) predictor, a pan-specific binding affinity predictorthat in principle covers all alleles, ranks SSAAAPFPL (SEQ ID NO: 21) asa strong binder to HLA-C*03:04 (23.2 nM, ranked 2nd for patient1-024-002), predicts weak binding of FVSTSDIKSM (SEQ ID NO: 22) toHLA-C*03:04 (943.4 nM, ranked 39th for patient 1-024-002) and weakbinding of YEHEDVKEA (SEQ ID NO: 20) to HLA-B*49:01 (3387.8 nM), butstronger binding to HLA-B*41:01 (208.9 nM, ranked 1th for patient1-038-001), which is also present in this patient but is not covered bythe model. Thus, of these three peptides, FVSTSDIKSM (SEQ ID NO: 22)would have been missed by binding affinity prediction, SSAAAPFPL (SEQ IDNO: 21) would have been captured, and the HLA restriction of YEHEDVKEA(SEQ ID NO: 20) is uncertain.

The remaining five peptides for which a peptide-specific T-cell responsewas deconvolved came from patients where the most probable presentingallele as determined by the model was also covered by MHCflurry 1.2.0.Of these five peptides, 4/5 had predicted binding affinities strongerthan the standard 500 nM threshold and ranked in the top 20, though withsomewhat lower ranks than the ranks from the model (peptides DENITTIQF(SEQ ID NO: 23), QDVSVQVER (SEQ ID NO: 24), EVADAATLTM (SEQ ID NO: 25),DTVEYPYTSF (SEQ ID NO: 26) were ranked 0, 4, 5, 7 by the modelrespectively vs 2, 14, 7, and 9 by MHCflurry). Peptide GTKKDVDVLK (SEQID NO: 27) was recognized by CD8 T-cells and ranked 1 by the model, buthad rank 70 and predicted binding affinity 2169 nM by MHCflurry.

Overall, 6/8 of the individually-recognized peptides that were rankedhighly by the full MS model also ranked highly using binding affinityprediction and had predicted binding affinity <500 nM, while 28 of theindividually-recognized peptides would have been missed if bindingaffinity prediction had been used instead of the full MS model.

XIV.I. Supplementary Table 4: MSD Cytokine Multiplex and ELISA Assays onELISpot Supernatants from NSCLC Neoantigen Peptides

Analytes detected in supernatants from positive ELiSpot (IFNgamma) wellsare shown for granzyme B (ELISA), TNFalpha, IL-2 and IL-5 (MSD). Valuesare shown as average pg/ml from technical replicates. Positive valuesare shown in italics. Granzyme B ELISA: Values ≥1.5-fold over DMSObackground were considered positive. U-Plex MSD assay: Values ≥1.5-foldover DMSO background were considered positive.

XIV.J. Supplementary Table 5: Neoantigen and Infectious Disease Epitopesin IVS Control Experiments

Details on tumor cell line neoantigen and viral peptides tested in IVScontrol experiments shown in FIGS. 19A-B. Key fields include source cellline or virus, peptide sequence, and predicted presenting HLA allele.

XIV.K. Data

The MS peptide dataset used to train and test the prediction model(FIGS. 14A-D) is available at the MassIVE Archive (massive.ucsd.edu),accession number MSV000082648. Neoantigen peptides tested by ELISpot(FIGS. 15A-C and 19A-B) are included with the manuscript (SupplementaryTables 3 and 5).

XV. Methods of Examples 8-10 XV.A. Mass Spectrometry XV.A.1. Specimens

Archived frozen tissue specimens for mass spectrometry analysis wereobtained from commercial sources, including BioServe (Beltsville, Md.),ProteoGenex (Culver City, Calif.), iSpecimen (Lexington, Mass.), andIndivumed (Hamburg, Germany). A subset of specimens was also collectedprospectively from patients at Hopital Marie Lannelongue (LePlessis-Robinson, France) under a research protocol approved by theComité de Protection des Personnes, Ile-de-France VII.

XV.A.2. HLA Immunoprecipitation

Isolation of HLA-peptide molecules was performed using establishedimmunoprecipitation (IP) methods after lysis and solubilization of thetissue sample^(97,124-126). Fresh frozen tissue was pulverized(CryoPrep; Covaris, Woburn, Mass.), lysis buffer (1% CHAPS. 20 mMTris-HCl, 150 mM NaCl, protease and phosphatase inhibitors, pH=8) wasadded to solubilize the tissue and the resultant solution wascentrifuged at 4 C for 2 hrs to pellet debris. The clarified lysate isused for the HLA specific IP. Immunoprecipitation was performed aspreviously described using the antibody W6/32¹²⁷. The lysate is added tothe antibody beads and rotated at 4 C overnight for theimmunoprecipitation. After immunoprecipitation, the beads were removedfrom the lysate. The IP beads were washed to remove non-specific bindingand the HLA/peptide complex was eluted from the beads with 2N aceticacid. The protein components were removed from the peptides using amolecular weight spin column. The resultant peptides were taken todryness by SpeedVac evaporation and stored at −20 C prior to MSanalysis.

XV.A.3. Peptide Sequencing

Dried peptides were reconstituted in HPLC buffer A and loaded onto aC-18 microcapillary HPLC column for gradient elution in to the massspectrometer. A gradient of 0-40% B (solvent A—0.1% formic acid, solventB— 0.1% formic acid in 80% acetonitrile) in 180 minutes was used toelute the peptides into the Fusion Lumos mass spectrometer (Thermo). MSIspectra of peptide mass/charge (m/z) were collected in the Orbitrapdetector with 120,000 resolution followed by 20 MS2 low resolution scanscollected in the either the Orbitrap or ion trap detector after HCDfragmentation of the selected ion. Selection of MS2 ions was performedusing data dependent acquisition mode and dynamic exclusion of 30seconds after MS2 selection of an ion. Automatic gain control (AGC) forMS1 scans was set to 4×105 and for MS2 scans was set to 1×104. Forsequencing HLA peptides, +1, +2 and +3 charge states can be selected forMS2 fragmentation.

MS2 spectra from each analysis were searched against a protein databaseusing Comet^(128,129) and the peptide identification were scored usingPercolator¹³⁰⁻¹³².

XV.B. Machine Learning XV.B.1. Data Encoding

For each sample, the training data points were all 8-11 mer (inclusive)peptides from the reference proteome that mapped to exactly one geneexpressed in the sample. The overall training dataset was formed byconcatenating the training datasets from each training sample. Lengths8-11 were chosen as this length range captures ˜95% of all HLA class Ipresented peptides; however, adding lengths 12-15 to the model could beaccomplished using the same methodology, at the cost of a modestincrease in computational demands. Peptides and flanking sequence werevectorized using a one-hot encoding scheme. Peptides of multiple lengths(8-11) were represented as fixed-length vectors by augmenting the aminoacid alphabet with a pad character and padding all peptides to themaximum length of 11. RNA abundance of the source protein of thetraining peptides was represented as the logarithm of the isoform-leveltranscripts per million (TPM) estimate obtained from RSEM¹³. For eachpeptide, the per-peptide TPM was computed as the sum of the per-isoformTPM estimates for each of the isoforms that contain the peptide.Peptides from genes expressed at 0 TPM were excluded from the trainingdata, and at test time, peptides from non-expressed genes are assigned aprobability of presentation of 0. Lastly, each peptide was assigned toan Ensembl protein family ID, and each unique Ensembl protein family IDcorresponded to a per-gene presentation propensity intercept (see nextsection).

XV.B.2. Specification of the Model Architecture

The full presentation model has the following functional form:

Pr(peptide i presented)=Σ_(k=1) ^(m) a _(k) ^(i) ·Pr(peptide i presentedby allele a),  (Equation 1)

where k indexes HLA alleles in the dataset, which run from 1 to m, anda_(k) ^(i) is an indicator variable whose value is 1 if allele k ispresent in the sample from which peptide i is derived and 0 otherwise.Note that for a given peptide i, all but at most 6 of the a_(k) ^(i)(the 6 corresponding to the HLA type of the sample of origin of peptidei) will be zero. The sum of probabilities is clipped at 1−ϵ, with ϵ=10⁻⁶for instance.

The per-allele probabilities of presentation are modeled as below:

Pr(peptide i presented by allelea)=sigmoid{NN_(a)(peptide_(i))+NN_(flanking)(flanking)+NN_(RNA)(log(TPM_(i)))+α_(sample(i))+β_(protein(i))},

where the variables have the following meanings: sigmoid is the sigmoid(aka expit) function, peptide_(i) is the onehot-encoded middle-paddedamino acid sequence of peptide i, NN_(α) is a neural network with linearlast-layer activation modeling the contribution of the peptide sequenceto the probability of presentation, flanking, is the onehot-encodedflanking sequence of peptide i in its source protein, NN_(flanking) is aneural network with linear last-layer activation modeling thecontribution of the flanking sequence to the probability ofpresentation, TPM_(i) is the expression of the source mRNAs of peptide iin TPM units, sample(i) is the sample (i.e., patient) of origin ofpeptide i, α_(sample(i)) is a per-sample intercept, protein(i) is thesource protein of peptide i, and β_(protein(i)) is a per-proteinintercept (aka the per-gene propensity of presentation).

For the models described in the results section, the component neuralnetworks have the following architectures:

-   -   Each of the NN_(a) is one output node of a one-hidden-layer        multi-layer-perceptron (MLP) with input dimension 231 (11        residues×21 possible characters per residue, including the pad        character), width 256, rectified linear unit (ReLU) activations        in the hidden layer, linear activation in the output layer, and        one output node per HLA allele a in the training dataset.    -   NN_(flanking) is a one-hidden-layer MLP with input dimension 210        (5 residues of N-terminal flanking sequence+5 residues of        C-terminal flanking sequence×21 possible characters per residue,        including the pad character), width 32, rectified linear unit        (ReLU) activations in the hidden layer and linear activation in        the output layer.    -   NN_(RNA) is a one-hidden-layer MLP with input dimension 1, width        16, rectified linear unit (ReLU) activations in the hidden layer        and linear activation in the output layer.

Note that some components of the model (e.g., NN_(a)) depend on aparticular HLA allele, but many components (NN_(flanking), NN_(RNA),α_(sample(i)), β_(protein(i))) do not. The former is referred to as“allele-interacting” and the latter as “allele-noninteracting”. Featuresto model as allele-interacting or noninteracting were chosen on thebasis of biological prior knowledge: the HLA allele sees the peptide, sothe peptide sequence should be modeled as allele-interacting, but noinformation about the source protein, RNA expression or flankingsequence is conveyed to the HLA molecule (as the peptide has beenseparated from its source protein by the time it encounters the HLA inthe endoplasmic reticulum), so these features should be modeled asallele-noninteracting. The model was implemented in Keras v2.0.4¹³⁴ andTheano v0.9.0¹³⁵.

The peptide MS model used the same deconvolution procedure as the fullMS model (Equation 1), but the per-allele probabilities of presentationwere generated using reduced per-allele models that consider onlypeptide sequence and HLA allele:

Pr(peptide i presented by allele a)=sigmoid{NN_(a)(peptide_(i))}.

The peptide MS model uses the same features as binding affinityprediction, but the weights of the model are trained on a different datatype (i.e., mass spectrometry data vs HLA-peptide binding affinitydata). Therefore, comparing the predictive performance of the peptide MSmodel to the full MS model reveals the contribution of non-peptidefeatures (i.e., RNA abundance, flanking sequence, gene ID) to theoverall predictive performance, and comparing the predictive performanceof the peptide MS model to the binding affinity models reveals theimportance of improved modeling of the peptide sequence to the overallpredictive performance.

XV.B.3. Train/Validate/Test Splits

We ensured that no peptides appeared in more than one of thetraining/validation/testing sets using the following procedure: first byremoving all peptides from the reference proteome that appear in morethan one protein, then by partitioning the proteome into blocks of 10adjacent peptides. Each block was assigned uniquely to the training,validation or testing sets. In this way, no peptide appears in more thanone of the training, validation on testing sets. The validation set wasused only for early stopping. The tumor sample test data in FIG. 14Arepresent test set peptides (i.e., peptides from the blocks of adjacentpeptides assigned uniquely to the test set) from five tumor samples thatwere held out of the training and validation sets entirely. Peptidesfrom the single-allele samples were included in the training data, butthe set of peptides (both presented and non-presented) incorporated intothe training and validation sets was disjoint from the set of peptidesused as test data in FIG. 14B.

XV.B.4. Model Training

For model training, all peptides were modeled as independent where theper-peptides loss is the negative Bernoulli log-likelihood loss function(aka log loss). Formally, the contribution of peptide i to the overallloss is

Loss(i)=−log(Bernoulli(y _(i) |Pr(peptide ipresented))),

where y_(i) is the label of peptide i; i.e., y_(i)=1 if peptide i ispresented and 0 otherwise, and Bernoulli(y|p) detnoes the Bemoullilikelihood of parameter p∈[0,1] given i.i.d. binary observation vectory. The model was trained by minimizing the loss function.

In order to reduce training time, the class balance was adjusted byremoving 90% of the negative-labeled training data at random, yieldingan overall training set class balance of one presented peptide per ˜2000non-presented peptides. Model weights were initialized using the Glorotuniform procedure61 and trained using the ADAM62 stochastic optimizerwith standard parameters on Nvidia Maxwell TITAN×GPUs. A validation setconsisting of 10% of the total data was used for early stopping. Themodel was evaluated on the validation set every quarter-epoch and modeltraining was stopped after the first quarter-epoch where the validationloss (i.e., the negative Bemoulli log-likelihood on the validation set)failed to decrease.

The full presentation model was an ensemble of 10 model replicates, witheach replicate trained independently on a shuffled copy of the sametraining data with a different random initialization of the modelweights for every model within the ensemble. At test time, predictionswere generated by taking the mean of the probabilities output by themodel replicates.

XV.B.5. Motif Logos

Motif logos were generated using the weblogolib Python API v3.5.0¹³⁸. Togenerate binding affinity logos, the mhc_ligand_full.csv file wasdownloaded from the Immune Epitope Database (IEDB⁸⁸) in July, 2017 andpeptides meeting the following criteria were retained: measurement innanomolar (nM) units, reference date after 2000, object type equal to“linear peptide” and all residues in the peptide drawn from thecanonical 20-letter amino acid alphabet. Logos were generated using thesubset of the filtered peptides with measured binding affinity below theconventional binding threshold of 500 nM. For alleles pair with too fewbinders in IEDB, logos were not generated. To generate logosrepresenting the learned presentation model, model predictions for2,000,000 random peptides were predicted for each allele and eachpeptide length. For each allele and each length, the logos weregenerated using the peptides ranked in the top 1% (i.e., the top 20,000)by the learned presentation model. Importantly, this binding affinitydata from IEDB was not used in model training or testing, but ratherused only for the comparison of motifs learned.

XV.B.6. Binding Affinity Prediction

We predicted peptide-MHC binding affinities using the bindingaffinity-only predictor from MHCflurry v1.2.0¹³⁹, an open-source,GPU-compatible HLA class I binding affinity predictor with performancecomparable to the NetMHC family of models. To combine binding affinitypredictions for a single peptide across multiple HLA alleles, theminimum binding affinity was selected. To combine binding affinitiesacross multiple peptides (i.e., in order to rank mutations spanned bymultiple mutated peptides as in FIG. 14C), the minimum binding affinityacross the peptides was selected. For RNA expression thresholding on theT-cell dataset, tumor-type matched RNA-seq data from TCGA to thresholdat TPM>1 was used. All of the original T-cell datasets were filtered onTPM>0 in the original publications, so the TCGA RNA-seq data to filteron TPM>0 was not used.

XV.B.7. Presentation Prediction

To combine probabilities of presentation for a single peptide acrossmultiple HLA alleles, the sum of the probabilities was identified, as inEquation 1. To combine probabilities of presentation across multiplepeptides (i.e., in order to rank mutations spanned by multiple peptidesas in FIG. 14C), the sum of the probabilities of presentation wasidentified. Probabilistically, if presentation of the peptides is viewedas i.i.d. Bemoulli random variables, the sum of the probabilitiescorresponds to the expected number of presented mutated peptides:

${{E\left\lbrack {\# \mspace{14mu} {presented}\mspace{14mu} {neoantigens}\mspace{14mu} {spanning}\mspace{14mu} {mutation}\mspace{14mu} i} \right\rbrack} = {\sum\limits_{j = 1}^{n_{i}}{\Pr \left\lbrack {{epitope}\mspace{14mu} j\mspace{14mu} {presented}} \right\rbrack}}},$

where Pr[epitope j presented] is obtained by applying the trainedpresentation model to epitope j, and n_(i) denotes the number of mutatedepitopes spanning mutation i. For example, for an SNV i distant from thetermini of its source gene, there are 8 spanning 8-mers, 9-spanning9-mers, 10 spanning 10-mers and 11 spanning 11-mers, for a total ofn_(i)=38 spanning mutated epitopes.

XV.C. Next Generation Seauencine XV.C.1. Specimens

For transcriptome analysis of the frozen resected tumors, RNA wasobtained from same tissue specimens (tumor or adjacent normal) as usedfor MS analyses. For neoantigen exome and transcriptome analysis inpatients on anti-PD1 therapy. DNA and RNA was obtained from archivalFFPE tumor biopsies. Adjacent normal, matched blood or PBMCs were usedto obtain normal DNA for normal exome and HLA typing.

XV.C.2. Nucleic Acid Extraction and Library Construction

Normal/germline DNA derived from blood were isolated using Qiagen DNeasycolumns (Hilden, Germany) following manufacturer recommended procedures.DNA and RNA from tissue specimens were isolated using Qiagen AllprepDNA/RNA isolation kits following manufacturer recommended procedures.The DNA and RNA were quantitated by Picogreen and Ribogreen Fluorescence(Molecular Probes), respectively specimens with >50 ng yield wereadvanced to library construction. DNA sequencing libraries weregenerated by acoustic shearing (Covaris, Woburn, Mass.) followed by DNAUltra II (NEB. Beverly, Mass.) library preparation kit following themanufacturers recommended protocols. Tumor RNA sequencing libraries weregenerated by heat fragmentation and library construction with RNA UltraII (NEB). The resulting libraries were quantitated by Picogreen(Molecular Probes).

XV.C.3. Whole Exome Capture

Exon enrichment for both DNA and RNA sequencing libraries was performedusing xGEN Whole Exome Panel (Integrated DNA Technologies). One to 1.5μg of normal DNA or tumor DNA or RNA-derived libraries were used asinput and allowed to hybridize for greater than 12 hours followed bystreptavidin purification. The captured libraries were minimallyamplified by PCR and quantitated by NEBNext Library Quant Kit (NEB).Captured libraries were pooled at equimolar concentrations and clusteredusing the c-bot (Illumina) and sequenced at 75 base paired-end on aHiSeq4000 (Illumina) to a target unique average coverage of >500× tumorexome, >100× normal exome, and >00M reads tumor transcriptome.

XV.C.4. Analysis

Exome reads (FFPE tumor and matched normals) were aligned to thereference human genome (hg38) using BWA-MEM¹⁴⁴ (v. 0.7.13-r1126).RNA-seq reads (FFPE and frozen tumor tissue samples) were aligned to thegenome and GENCODE transcripts (v. 25) using STAR (v. 2.5.1b). RNAexpression was quantified using RSEM¹³³ (v. 1.2.31) with the samereference transcripts. Picard (v. 2.7.1) was used to mark duplicatealignments and calculate alignment metrics. For FFPE tumor samplesfollowing base quality score recalibration with GATK¹⁴⁵ (v. 3.5-0),substitution and short indel variants were determined using pairedtumor-normal exomes with FreeBayes¹⁴⁶ (1.0.2). Filters included allelefrequency >4%; median base quality >25, minimum mapping quality ofsupporting reads 30, and alternate read count in normal ⇐2 withsufficient coverage obtained. Variants must also be detected on bothstrands. Somatic variants occurring in repetitive regions were excluded.Translation and annotation were performed with snpEff¹⁴⁷ (v. 4.2) usingRefSeq transcripts. Non-synonymous, non-stop variants verified in tumorRNA alignments were advanced to neoantigen prediction. Optitype¹⁴⁸ 1.3.1was used to generate HLA types.

XV.C.5. FIGS. 19A-B: Tumor Cell Lines and Matched Normals for IVSControl Experiments

Tumor cell lines H128, H122, H2009, H2126, Colo829 and their normaldonor matched control cell lines BL128, BL2122, BL2009, BL2126 andColo829BL were all purchased from ATCC (Manassas, Va.) were grown to10⁸³-10⁸⁴ cells per seller's instructions then snap frozen for nucleicacid extraction and sequencing. NGS processing was performed generallyas described above, except that MuTect¹⁴⁹ (3.1-0) was used forsubstitution mutation detection only. Peptides used in the IVS controlassays are listed in Supplementary Table 5.

XV.D. Class II Model Proof-of-Concept

We evaluated whether the prediction model disclosed herein can also beapplied to class II HLA peptide presentation. To do this, publishedclass II mass spectrometry data was obtained for two cell lines, each ofwhich expressed a single HLA class I allele. One cell line expressedHLA-DRB1*15:01 and the other expressed HLA-DRB5*01:01¹⁵⁰. These two celllines were used for training data. For test data, class II massspectrometry data was obtained from a separate cell line expressing bothHLA-DRB*15:01 and HLA-DRB5*01:01.¹⁵¹ RNA sequencing data was notavailable either the training or testing cell lines, thereforeRNA-sequencing data from a different B-cell line, B721.221⁹², wassubstituted.

The peptide sets were split into training, validation and testing setsusing the same procedure as for the HLA class I data, except that forthe class II data peptides with lengths between 9 and 20 were included.The training data included 330 peptides presented by HLA-DRB1*15:01, and103 peptides presented by HLA-DRB5*01:01. The test dataset included 223peptides presented by either HLA-DRB1*15:01 or HLA-DRB5*01:01 along with4708 non-presented peptides.

We trained an ensemble of 10 models on the training dataset to predictHLA class II peptide presentation. The architecture and trainingprocedures for these models were identical to those used to predictclass I presentation, with the exception that class II models took asinput peptides sequences one hot-encoded and zero-padded to length 20rather than 11.

FIG. 23 compares the predictive performance of the the “MS Model,”“NetMHCIIpan rank”: NetMHCIIpan 3.1⁷⁷, taking the lowest NetMHCIIpanpercentile rank across HLA-DRB1*15:01 and HLA-DRB5*01:01, and“NetMHCIIpan nM”: NetMHCIIpan 3.1, taking the strongest affinity in nMunits across HLA-DRB1*15:01 and HLA-DRB5*01:01, at ranking the peptidesin the HLA-DRB1*15:01/HLA-DRB5*01:01 test dataset. The “MS Model” is theMHC class II presentation prediction model disclosed herein.

Specifically. FIG. 23 depicts receiver operating characteristic (ROC)curves and the area under the ROC curve AUC (panel A) and AUC_(0.1)(panel B) statistics for these ranking methods. AUC_(0.1) is AUC between0 and 0.1FPR*10, commonly considered in the epitope prediction field¹⁹.The NetMHCIIpan nM and rank methods performed similarly. The MS modelperformed best, significantly exceeding performance of the comparatormethods, particularly in the critical high-specificity region of the ROCcurve (AUC_(0.1)0.41 vs. 0.27).

XVI. Example 11: Sequencing TCRs of Neoantigen-Specific Memory T-Cellsfrom Peripheral Blood of a NSCLC Patient

FIG. 24 depicts a method for sequencing TCRs of neoantigen-specificmemory T-cells from the peripheral blood of a NSCLC patient. Peripheralblood mononuclear cells (PBMCs) from NSCLC patient CU04 (described abovewith regard to FIGS. 15A-22) were collected after ELISpot incubation.Specifically, as discussed above, the in vitro expanded PBMCs from 2visits of patient CU04 were stimulated in IFN-gamma ELISpot with theCU04-specific individual neoantigen peptides (FIG. 21C), with theCU04-specific neoantigen peptide pool (FIG. 21C), and with DMSO negativecontrol (FIG. 22). Following incubation and prior to addition ofdetection antibody, the PBMCs were transferred to a new culture plateand maintained in an incubator during completion of the ELISpot assay.Positive (responsive) wells were identified based on ELISpot results. Asshown in FIG. 21, the positive wells identified include the wellsstimulated with CU04-specific individual neoantigen peptide 8 and thewells simulated with the CU04-specific neoantigen peptide pool. Cellsfrom these positive wells and negative control (DMSO) wells werecombined and stained for CD137 with magnetically-labelled antibodies forenrichment using Miltenyi magnetic isolation columns.

CD137-enriched and -depleted T-cell fractions isolated and expanded asdescribed above were sequenced using 10× Genomics single cell resolutionpaired immune TCR profiling approach. Specifically, live T cells werepartitioned into single cell emulsions for subsequent single cell cDNAgeneration and full-length TCR profiling (5′ UTR through constant region—ensuring alpha and beta pairing). One approach utilizes a molecularlybarcoded template switching oligo at the 5′end of the transcript, asecond approach utilizes a molecularly barcoded constant region oligo atthe 3′ end, and a third approach couples an RNA polymerase promoter toeither the 5′ or 3′ end of a TCR All of these approaches enable theidentification and deconvolution of alpha and beta TCR pairs at thesingle-cell level. The resulting barcoded cDNA transcripts underwent anoptimized enzymatic and library construction workflow to reduce bias andensure accurate representation of clonotypes within the pool of cells.Libraries were sequenced on Illumina's MiSeq or HiSeq4000 instruments(paired-end 150 cycles) for a target sequencing depth of about five tofifty thousand reads per cell. The resulting TCR nucleic acid sequencesare depicted in Supplementary Table 6. The presence of the TCRa and TCRbchains described in Supplementary Table 6 were confirmed by anorthogonal anchor-PCR based TCR sequencing approach (Archer). Thisparticular approach has the advantage of using limited cell numbers asinput and fewer enzymatic manipulations when compared to the 10×Genomics based TCR sequencing.

Sequencing outputs were analyzed using the 10× software and custombioinformatics pipelines to identify T-cell receptor (TCR) alpha andbeta chain pairs as also shown in Supplementary Table 6. Supplementarytable 6 further lists the alpha and beta variable (V), joining (J),constant (C), and beta diversity (D) regions, and CDR3 amino acidsequence of the most prevalent TCR clonotypes. Clonotypes were definedas alpha, beta chain pairs of unique CDR3 amino acid sequences.Clonotypes were filtered for single alpha and single beta chain pairspresent at frequency above 2 cells to yield the final list of clonotypesper target peptide in patient CU04 (Supplementary Table 6).

In summary, using the method described above with regard to FIG. 24,memory CD8+ T-cells from the peripheral blood of patient CU04, that areneoantigen-specific to patient CU04's tumor neoantigens identified asdiscussed above with regard to Example 10 in Section XIV., wereidentified. The TCRs of these identified neoantigen-specific T-cellswere sequenced. And furthermore, sequenced TCRs that areneoantigen-specific to patient CU04's tumor neoantigens as identified bythe above presentation models, were identified.

XVI. Example 12: Use of Neoantigen-Specific Memory T-Cells for T-CellTherapy

After T-cells and/or TCRs that are neoantigen-specific to neoantigenspresented by a patient's tumor are identified, these identifiedneoantigen-specific T-cells and/or TCRs can be used for T-cell therapyin the patient. Specifically, these identified neoantigen-specificT-cells and/or TCRs can be used to produce a therapeutic quantity ofneoantigen-specific T-cells for infusion into a patient during T-celltherapy. Two methods for producing a therapeutic quantity of neoantigenspecific T-cells for use in T-cell therapy in a patient are discussedherein in Sections XVII.A. and XVII.B. The first method comprisesexpanding the identified neoantigen-specific T-cells from a patientsample (Section XVII.A.). The second method comprises sequencing theTCRs of the identified neoantigen-specific T-cells and cloning thesequenced TCRs into new T-cells (Section XVII.B.). Alternative methodsfor producing neoantigen specific T-cells for use in T-cell therapy thatare not explicitly mentioned herein can also be used to produce atherapeutic quantity of neoantigen specific T-cells for use in T-celltherapy. Once the neoantigen-specific T-cells are obtained via one ormore of these methods, these neoantigen-specific T-cells may be infusedinto the patient for T-cell therapy.

XVII.A. Identification and Expansion of Neoantigen-Specific MemoryT-Cells from a Patient Sample for T-Cell Therapy

A first method for producing a therapeutic quantity of neoantigenspecific T-cells for use in T-cell therapy in a patient comprisesexpanding identified neoantigen-specific T-cells from a patient sample.

Specifically, to expand neoantigen-specific T-cells to a therapeuticquantity for use in T-cell therapy in a patient, a set of neoantigenpeptides that are most likely to be presented by a patient's cancercells are identified using the presentation models as described above.Additionally, a patient sample containing T-cells is obtained from thepatient. The patient sample may comprise the patient's peripheral blood,tumor-infiltrating lymphocytes (TIL), or lymph node cells.

In embodiments in which the patient sample comprises the patient'speripheral blood, the following methods may be used to expandneoantigen-specific T-cells to a therapeutic quantity. In oneembodiment, priming may be performed. In another embodiment,already-activated T-cells may be identified using one or more of themethods described above. In another embodiment, both priming andidentification of already-activated T-cells may be performed. Theadvantage to both priming and identifying already-activated T-cells isto maximize the number of specificities represented. The disadvantageboth priming and identifying already-activated T-cells is that thisapproach is difficult and time-consuming. In another embodiment,neoantigen-specific cells that are not necessarily activated may beisolated. In such embodiments, antigen-specific or non-specificexpansion of these neoantigen-specific cells may also be performed.Following collection of these primed T-cells, the primed T-cells can besubjected to rapid expansion protocol. For example, in some embodiments,the primed T-cells can be subjected to the Rosenberg rapid expansionprotocol (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2978753/,https://www.ncbi.nlm.nih.gov/pmc/particles/PMC2305721/)^(153, 154).

In embodiments in which the patient sample comprises the patient's TIL,the following methods may be used to expand neoantigen-specific T-cellsto a therapeutic quantity. In one embodiment, neoantigen-specific TILcan be tetramer/multimer sorted ex vivo, and then the sorted TIL can besubjected to a rapid expansion protocol as described above. In anotherembodiment, neoantigen-nonspecific expansion of the TIL may beperformed, then neoantigen-specific TIL may be tetramer sorted, and thenthe sorted TIL can be subjected to a rapid expansion protocol asdescribed above. In another embodiment, antigen-specific culturing maybe performed prior to subjecting the TIL to the rapid expansionprotocol. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4607110/,https://onlinelibrary.wiley.com/doi/pdf/10.1002/eii.201545849)^(155, 156).

In some embodiments, the Rosenberg rapid expansion protocol may bemodified. For example, anti-PD1 and/or anti-41BB may be added to the TILculture to simulate more rapid expansion.(htps://jitc.biomedcentral.com/articles/10.1186/s40425-016-064-7)¹⁵⁷.

XVII.B. Identification of Neoantigen-Specific T Cells, Sequencing TCRsof Identified Neoantigen-Specific T Cells, and Cloning of Sequenced TCRsinto New T-Cells

A second method for producing a therapeutic quantity of neoantigenspecific T-cells for use in T-cell therapy in a patient comprisesidentifying neoantigen-specific T-cells from a patient sample,sequencing the TCRs of the identified neoantigen-specific T-cells, andcloning the sequenced TCRs into new T-cells.

First, neoantigen-specific T-cells are identified from a patient sample,and the TCRs of the identified neoantigen-specific T-cells aresequenced. The patient sample from which T cells can be isolated maycomprise one or more of blood, lymph nodes, or tumors. Morespecifically, the patient sample from which T cells can be isolated maycomprise one or more of peripheral blood mononuclear cells (PBMCs),tumor-infiltrating cells (TILs), dissociated tumor cells (DTCs), invitro primed T cells, and/or cells isolated from lymph nodes. Thesecells may be fresh and/or frozen. The PBMCs and the in vitro primed Tcells may be obtained from cancer patients and/or healthy subjects.

After the patient sample is obtained, the sample may be expanded and/orprimed. Various methods may be implemented to expand and prime thepatient sample. In one embodiment, fresh and/or frozen PBMCs may besimulated in the presence of peptides or tandem mini-genes. In anotherembodiment, fresh and/or frozen isolated T-cells may be simulated andprimed with antigen-presenting cells (APCs) in the presence of peptidesor tandem mini-genes. Examples of APCs include B-cells, monocytes,dendritic cells, macrophages or artificial antigen presenting cells(such as cells or beads presenting relevant HLA and co-stimulatorymolecules, reviewed inhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2929753). In anotherembodiment, PBMCs, TILs, and/or isolated T-cells may be stimulated inthe presence of cytokines (e.g., IL-2, IL-7, and/or IL-15). In anotherembodiment, TILs and/or isolated T-cells can be stimulated in thepresence of maximal stimulus, cytokine(s), and/or feeder cells. In suchembodiments, T cells can be isolated by activation markers and/ormultimers (e.g., tetramers). In another embodiment, TILs and/or isolatedT cells can be stimulated with stimulatory and/or co-stimulatory markers(e.g., CD3 antibodies, CD28 antibodies, and/or beads (e.g., DynaBeads).In another embodiment, DTCs can be expanded using a rapid expansionprotocol on feeder cells at high dose of IL-2 in rich media.

Then, neoantigen-specific T cells are identified and isolated. In someembodiments, T cells are isolated from a patient sample ex vivo withoutprior expansion. In one embodiment, the methods described above withregard to Section XVI, may be used to identify neoantigen-specific Tcells from a patient sample. In an alternative embodiment, isolation iscarried out by enrichment for a particular cell population by positiveselection, or depletion of a particular cell population, by negativeselection. In some embodiments, positive or negative selection isaccomplished by incubating cells with one or more antibodies or otherbinding agent that specifically bind to one or more surface markersexpressed or expressed (marker+) at a relatively higher level(marker^(high)) on the positively or negatively selected cells,respectively.

In some embodiments, T cells are separated from a PBMC sample bynegative selection of markers expressed on non-T cells, such as B cells,monocytes, or other white blood cells, such as CD14. In some aspects, aCD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+cytotoxic T-cells. Such CD4+ and CD8+ populations can be further sortedinto sub-populations by positive or negative selection for markersexpressed or expressed to a relatively higher degree on one or morenaive, memory, and/or effector T-cell subpopulations.

In some embodiments, CD8+ cells are further enriched for or depleted ofnaive, central memory, effector memory, and/or central memory stemcells, such as by positive or negative selection based on surfaceantigens associated with the respective subpopulation. In someembodiments, enrichment for central memory T (TCM) cells is carried outto increase efficacy, such as to improve long-term survival, expansion,and/or engraftment following administration, which in some aspects isparticularly robust in such sub-populations. See Terakura et al. (2012)Blood. 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In someembodiments, combining TCM-enriched CD8+ T-cells and CD4+ T-cellsfurther enhances efficacy.

In embodiments, memory T cells are present in both CD62L+ and CD62L−subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched foror depleted of CD62L-CD8+ and/or CD62L+CD8+ fractions, such as usinganti-CD8 and anti-CD62L antibodies.

In some embodiments, the enrichment for central memory T (TCM) cells isbased on positive or high surface expression of CD45RO, CD62L, CCR7,CD28, CD3, and/or CD 127; in some aspects, it is based on negativeselection for cells expressing or highly expressing CD45RA and/orgranzyme B. In some aspects, isolation of a CD8+ population enriched forTCM cells is carried out by depletion of cells expressing CD4, CD14,CD45RA, and positive selection or enrichment for cells expressing CD62L.In one aspect, enrichment for central memory T (TCM) cells is carriedout starting with a negative fraction of cells selected based on CD4expression, which is subjected to a negative selection based onexpression of CD14 and CD45RA, and a positive selection based on CD62L.Such selections in some aspects are carried out simultaneously and inother aspects are carried out sequentially, in either order. In someaspects, the same CD4 expression-based selection step used in preparingthe CD8+ cell population or subpopulation, also is used to generate theCD4+ cell population or sub-population, such that both the positive andnegative fractions from the CD4-based separation are retained and usedin subsequent steps of the methods, optionally following one or morefurther positive or negative selection steps.

In a particular example, a sample of PBMCs or other white blood cellsample is subjected to selection of CD4+ cells, where both the negativeand positive fractions are retained. The negative fraction then issubjected to negative selection based on expression of CD14 and CD45RAor ROR1, and positive selection based on a marker characteristic ofcentral memory T-cells, such as CD62L or CCR7, where the positive andnegative selections are carried out in either order.

CD4+T helper cells are sorted into naive, central memory, and effectorcells by identifying cell populations that have cell surface antigens.CD4+ lymphocytes can be obtained by standard methods. In someembodiments, naive CD4+T lymphocytes are CD45RO−, CD45RA+, CD62L+, CD4+T-cells. In some embodiments, central memory CD4+ cells are CD62L+ andCD45RO+. In some embodiments, effector CD4+ cells are CD62L− andCD45RO−.

In one example, to enrich for CD4+ cells by negative selection, amonoclonal antibody cocktail typically includes antibodies to CD14,CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody orbinding partner is bound to a solid support or matrix, such as amagnetic bead or paramagnetic bead, to allow for separation of cells forpositive and/or negative selection. For example, in some embodiments,the cells and cell populations are separated or isolated usingimmune-magnetic (or affinity-magnetic) separation techniques (reviewedin Methods in Molecular Medicine, vol. 58: Metastasis ResearchProtocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Editedby: S. A. Brooks and U. Schumacher Humana Press Inc., Totowa, N.J.).

In some aspects, the sample or composition of cells to be separated isincubated with small, magnetizable or magnetically responsive material,such as magnetically responsive particles or microparticles, such asparamagnetic beads (e.g., such as Dynabeads or MACS beads). Themagnetically responsive material, e.g., particle, generally is directlyor indirectly attached to a binding partner, e.g., an antibody, thatspecifically binds to a molecule, e.g., surface marker, present on thecell, cells, or population of cells that it is desired to separate,e.g., that it is desired to negatively or positively select.

In some embodiments, the magnetic particle or bead comprises amagnetically responsive material bound to a specific binding member,such as an antibody or other binding partner. There are many well-knownmagnetically responsive materials used in magnetic separation methods.Suitable magnetic particles include those described in Molday, U.S. Pat.No. 4,452,773, and in European Patent Specification EP 452342 B, whichare hereby incorporated by reference. Colloidal sized particles, such asthose described in Owen U.S. Pat. No. 4,795,698, and Liberti et al.,U.S. Pat. No. 5,200,084 are other examples.

The incubation generally is carried out under conditions whereby theantibodies or binding partners, or molecules, such as secondaryantibodies or other reagents, which specifically bind to such antibodiesor binding partners, which are attached to the magnetic particle orbead, specifically bind to cell surface molecules if present on cellswithin the sample.

In some aspects, the sample is placed in a magnetic field, and thosecells having magnetically responsive or magnetizable particles attachedthereto will be attracted to the magnet and separated from the unlabeledcells. For positive selection, cells that are attracted to the magnetare retained; for negative selection, cells that are not attracted(unlabeled cells) are retained. In some aspects, a combination ofpositive and negative selection is performed during the same selectionstep, where the positive and negative fractions are retained and furtherprocessed or subject to further separation steps.

In certain embodiments, the magnetically responsive particles are coatedin primary antibodies or other binding partners, secondary antibodies,lectins, enzymes, or streptavidin. In certain embodiments, the magneticparticles are attached to cells via a coating of primary antibodiesspecific for one or more markers. In certain embodiments, the cells,rather than the beads, are labeled with a primary antibody or bindingpartner, and then cell-type specific secondary antibody- or otherbinding partner (e.g., streptavidin)-coated magnetic particles, areadded. In certain embodiments, streptavidin-coated magnetic particlesare used in conjunction with biotinylated primary or secondaryantibodies.

In some embodiments, the magnetically responsive particles are leftattached to the cells that are to be subsequently incubated, culturedand/or engineered; in some aspects, the particles are left attached tothe cells for administration to a patient. In some embodiments, themagnetizable or magnetically responsive particles are removed from thecells. Methods for removing magnetizable particles from cells are knownand include, e.g., the use of competing non-labeled antibodies,magnetizable particles or antibodies conjugated to cleavable linkers,etc. In some embodiments, the magnetizable particles are biodegradable.

In some embodiments, the affinity-based selection is viamagnetic-activated cell sorting (MACS) (Miltenyi Biotech, Auburn,Calif.). Magnetic Activated Cell Sorting (MACS) systems are capable ofhigh-purity selection of cells having magnetized particles attachedthereto. In certain embodiments, MACS operates in a mode wherein thenon-target and target species are sequentially eluted after theapplication of the external magnetic field. That is, the cells attachedto magnetized particles are held in place while the unattached speciesare eluted. Then, after this first elution step is completed, thespecies that were trapped in the magnetic field and were prevented frombeing eluted are freed in some manner such that they can be eluted andrecovered. In certain embodiments, the non-large T cells are labelledand depleted from the heterogeneous population of cells.

In certain embodiments, the isolation or separation is carried out usinga system, device, or apparatus that carries out one or more of theisolation, cell preparation, separation, processing, incubation,culture, and/or formulation steps of the methods. In some aspects, thesystem is used to carry out each of these steps in a closed or sterileenvironment, for example, to minimize error, user handling and/orcontamination. In one example, the system is a system as described inInternational Patent Application, Publication Number WO2009/072003, orUS 20110003380 A1.

In some embodiments, the system or apparatus carries out one or more,e.g., all, of the isolation, processing, engineering, and formulationsteps in an integrated or self-contained system, and/or in an automatedor programmable fashion. In some aspects, the system or apparatusincludes a computer and/or computer program in communication with thesystem or apparatus, which allows a user to program, control, assess theoutcome of, and/or adjust various aspects of the processing, isolation,engineering, and formulation steps.

In some aspects, the separation and/or other steps is carried out usingCliniMACS system (Miltenyi Biotic), for example, for automatedseparation of cells on a clinical-scale level in a closed and sterilesystem. Components can include an integrated microcomputer, magneticseparation unit, peristaltic pump, and various pinch valves. Theintegrated computer in some aspects controls all components of theinstrument and directs the system to perform repeated procedures in astandardized sequence. The magnetic separation unit in some aspectsincludes a movable permanent magnet and a holder for the selectioncolumn. The peristaltic pump controls the flow rate throughout thetubing set and, together with the pinch valves, ensures the controlledflow of buffer through the system and continual suspension of cells.

The CliniMACS system in some aspects uses antibody-coupled magnetizableparticles that are supplied in a sterile, non-pyrogenic solution. Insome embodiments, after labelling of cells with magnetic particles thecells are washed to remove excess particles. A cell preparation bag isthen connected to the tubing set, which in turn is connected to a bagcontaining buffer and a cell collection bag. The tubing set consists ofpre-assembled sterile tubing, including a pre-column and a separationcolumn, and are for single use only. After initiation of the separationprogram, the system automatically applies the cell sample onto theseparation column. Labelled cells are retained within the column, whileunlabeled cells are removed by a series of washing steps. In someembodiments, the cell populations for use with the methods describedherein are unlabeled and are not retained in the column. In someembodiments, the cell populations for use with the methods describedherein are labeled and are retained in the column. In some embodiments,the cell populations for use with the methods described herein areeluted from the column after removal of the magnetic field, and arecollected within the cell collection bag.

In certain embodiments, separation and/or other steps are carried outusing the CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACSProdigy system in some aspects is equipped with a cell processing unitythat permits automated washing and fractionation of cells bycentrifugation. The CliniMACS Prodigy system can also include an onboardcamera and image recognition software that determines the optimal cellfractionation endpoint by discerning the macroscopic layers of thesource cell product. For example, peripheral blood may be automaticallyseparated into erythrocytes, white blood cells and plasma layers. TheCliniMACS Prodigy system can also include an integrated cell cultivationchamber which accomplishes cell culture protocols such as, e.g., celldifferentiation and expansion, antigen loading, and long-term cellculture. Input ports can allow for the sterile removal and replenishmentof media and cells can be monitored using an integrated microscope. See,e.g., Klebanoff et. al., (2012) J Immunother. 35(9): 651-660, Terakuraet al. (2012) Blood. 1:72-82, and Wang et al. (2012) J Immunother.35(9):689-701.

In some embodiments, a cell population described herein is collected andenriched (or depleted) via flow cytometry, in which cells stained formultiple cell surface markers are carried in a fluidic stream. In someembodiments, a cell population described herein is collected andenriched (or depleted) via preparative scale (FACS)-sorting. In certainembodiments, a cell population described herein is collected andenriched (or depleted) by use of microelectromechanical systems (MEMS)chips in combination with a FACS-based detection system (see, e.g., WO2010033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al.(2008) J Biophoton. 1(5):355-376. In both cases, cells can be labeledwith multiple markers, allowing for the isolation of well-defined T-cellsubsets at high purity.

In some embodiments, the antibodies or binding partners are labeled withone or more detectable marker, to facilitate separation for positiveand/or negative selection. For example, separation may be based onbinding to fluorescently labeled antibodies. In some examples,separation of cells based on binding of antibodies or other bindingpartners specific for one or more cell surface markers are carried in afluidic stream, such as by fluorescence-activated cell sorting (FACS),including preparative scale (FACS) and/or microelectromechanical systems(MEMS) chips, e.g., in combination with a flow-cytometric detectionsystem. Such methods allow for positive and negative selection based onmultiple markers simultaneously.

In some embodiments, the preparation methods include steps for freezing,e.g., cryopreserving, the cells, either before or after isolation,incubation, and/or engineering. In some embodiments, the freeze andsubsequent thaw step removes granulocytes and, to some extent, monocytesin the cell population. In some embodiments, the cells are suspended ina freezing solution, e.g., following a washing step to remove plasma andplatelets. Any of a variety of known freezing solutions and parametersin some aspects may be used. One example involves using PBS containing20% DMSO and 8% human serum albumin (HSA), or other suitable cellfreezing media. This can then be diluted 1:1 with media so that thefinal concentration of DMSO and HSA are 10% and 4%, respectively. Otherexamples include Cryostor®, CTL-CryoM ABC freezing media, and the like.The cells are then frozen to −80 degrees C. at a rate of 1 degree perminute and stored in the vapor phase of a liquid nitrogen storage tank.

In some embodiments, the provided methods include cultivation,incubation, culture, and/or genetic engineering steps. For example, insome embodiments, provided are methods for incubating and/or engineeringthe depleted cell populations and culture-initiating compositions.

Thus, in some embodiments, the cell populations are incubated in aculture-initiating composition. The incubation and/or engineering may becarried out in a culture vessel, such as a unit, chamber, well, column,tube, tubing set, valve, vial, culture dish, bag, or other container forculture or cultivating cells.

In some embodiments, the cells are incubated and/or cultured prior to orin connection with genetic engineering. The incubation steps can includeculture, cultivation, stimulation, activation, and/or propagation. Insome embodiments, the compositions or cells are incubated in thepresence of stimulating conditions or a stimulatory agent. Suchconditions include those designed to induce proliferation, expansion,activation, and/or survival of cells in the population, to mimic antigenexposure, and/or to prime the cells for genetic engineering, such as forthe introduction of a recombinant antigen receptor.

The conditions can include one or more of particular media, temperature,oxygen content, carbon dioxide content, time, agents, e.g., nutrients,amino acids, antibiotics, ions, and/or stimulatory factors, such ascytokines, chemokines, antigens, binding partners, fusion proteins,recombinant soluble receptors, and any other agents designed to activatethe cells.

In some embodiments, the stimulating conditions or agents include one ormore agent, e.g., ligand, which is capable of activating anintracellular signaling domain of a TCR complex. In some aspects, theagent turns on or initiates TCR/CD3 intracellular signaling cascade in aT-cell. Such agents can include antibodies, such as those specific for aTCR component and/or costimulatory receptor, e.g., anti-CD3, anti-CD28,for example, bound to solid support such as a bead, and/or one or morecytokines. Optionally, the expansion method may further comprise thestep of adding anti-CD3 and/or anti CD28 antibody to the culture medium(e.g., at a concentration of at least about 0.5 ng/ml). In someembodiments, the stimulating agents include IL-2 and/or IL-15, forexample, an IL-2 concentration of at least about 10 units/mL.

In some aspects, incubation is carried out in accordance with techniquessuch as those described in U.S. Pat. No. 6,040,177 to Riddell et al.,Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al.(2012) Blood. 1:72-82, and/or Wang et al. (2012) J Immunother.35(9):689-701.

In some embodiments, the T-cells are expanded by adding to theculture-initiating composition feeder cells, such as non-dividingperipheral blood mononuclear cells (PBMC), (e.g., such that theresulting population of cells contains at least about 5, 10, 20, or 40or more PBMC feeder cells for each T lymphocyte in the initialpopulation to be expanded); and incubating the culture (e.g. for a timesufficient to expand the numbers of T-cells). In some aspects, thenon-dividing feeder cells can comprise gamma-irradiated PBMC feedercells. In some embodiments, the PBMC are irradiated with gamma rays inthe range of about 3000 to 3600 rads to prevent cell division. In someembodiments, the PBMC feeder cells are inactivated with Mytomicin C. Insome aspects, the feeder cells are added to culture medium prior to theaddition of the populations of T-cells.

In some embodiments, the stimulating conditions include temperaturesuitable for the growth of human T lymphocytes, for example, at leastabout 25 degrees Celsius, generally at least about 30 degrees, andgenerally at or about 37 degrees Celsius. Optionally, the incubation mayfurther comprise adding non-dividing EBV-transformed lymphoblastoidcells (LCL) as feeder cells. LCL can be irradiated with gamma rays inthe range of about 6000 to 10,000 rads. The LCL feeder cells in someaspects is provided in any suitable amount, such as a ratio of LCLfeeder cells to initial T lymphocytes of at least about 10:1.

In embodiments, antigen-specific T-cells, such as antigen-specific CD4+and/or CD8+ T-cells, are obtained by stimulating naive or antigenspecific T lymphocytes with antigen. For example, antigen-specificT-cell lines or clones can be generated to cytomegalovirus antigens byisolating T-cells from infected subjects and stimulating the cells invitro with the same antigen.

In some embodiments, neoantigen-specific T-cells are identified and/orisolated following stimulation with a functional assay (e.g., ELISpot).In some embodiments, neoantigen-specific T-cells are isolated by sortingpolyfunctional cells by intracellular cytokine staining. In someembodiments, neoantigen-specific T-cells are identified and/or isolatedusing activation markers (e.g., CD137, CD38, CD38/HLA-DRdouble-positive, and/or CD69). In some embodiments, neoantigen-specificCD8+, natural killer T-cells, memory T-cells, and/or CD4+ T-cells areidentified and/or isolated using class I or class II multimers and/oractivation markers. In some embodiments, neoantigen-specific CD8+ and/orCD4+ T-cells are identified and/or isolated using memory markers (e.g.,CD45RA, CD45RO, CCR7, CD27, and/or CD62L). In some embodiments,proliferating cells are identified and/or isolated. In some embodiments,activated T-cells are identified and/or isolated.

After identification of neoantigen-specific T-cells from a patientsample, the neoantigen-specific TCRs of the identifiedneoantigen-specific T-cells are sequenced. To sequence aneoantigen-specific TCR, the TCR must first be identified. One method ofidentifying a neoantigen-specific TCR of a T-cell can include contactingthe T-cell with an HLA-multimer (e.g., a tetramer) comprising at leastone neoantigen; and identifying the TCR via binding between theHLA-multimer and the TCR. Another method of identifying aneoantigen-specific TCR can include obtaining one or more T-cellscomprising the TCR; activating the one or more T-cells with at least oneneoantigen presented on at least one antigen presenting cell (APC); andidentifying the TCR via selection of one or more cells activated byinteraction with at least one neoantigen.

After identification of the neoantigen-specific TCR the TCR can besequenced. In one embodiment, the methods described above with regard toSection XVI, may be used to sequence TCRs. In another embodiment. TCRaand TCRb of a TCR can be bulk-sequenced and then paired based onfrequency. In another embodiment, TCRs can be sequenced and paired usingthe method of Howie et al., Science Translational Medicine 2015 (doi:10.1126/scitranslmed.aac5624). In another embodiment, TCRs can besequenced and paired using the method of Han et al., Nat Biotech 2014(PMID 24952902, doi 10.1038/nbt.2938). In another embodiment, paired TCRsequences can be obtained using the method described byhttps://www.biorxiv.org/content/early/2017/05/05/134841 andhttps://patents.google.com;/patent/US20160244825A1/.^(158, 159)

In another embodiment, clonal populations of T cells can be produced bylimiting dilution, and then the TCRa and TCRb of the clonal populationsof T cells can be sequenced. In yet another embodiment, T-cells can besorted onto a plate with wells such that there is one T cell per well,and then the TCRa and TCRb of each T cell in each well can be sequencedand paired.

Next, after neoantigen-specific T-cells are identified from a patientsample and the TCRs of the identified neoantigen-specific T-cells aresequenced, the sequenced TCRs are cloned into new T-cells. These clonedT-cells contain neoantigen-specific receptors, e.g., containextracellular domains including TCRs. Also provided are populations ofsuch cells, and compositions containing such cells. In some embodiments,compositions or populations are enriched for such cells, such as inwhich cells expressing the TCRs make up at least 1, 5, 10, 20, 30, 40,50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or more than 99percent of the total cells in the composition or cells of a certain typesuch as T-cells or CD8+ or CD4+ cells. In some embodiments, acomposition comprises at least one cell containing a TCR disclosedherein. Among the compositions are pharmaceutical compositions andformulations for administration, such as for adoptive cell therapy. Alsoprovided are therapeutic methods for administering the cells andcompositions to subjects, e.g., patients.

Thus also provided are genetically engineered cells expressing TCR(s).The cells generally are eukaryotic cells, such as mammalian cells, andtypically are human cells. In some embodiments, the cells are derivedfrom the blood, bone marrow, lymph, or lymphoid organs, are cells of theimmune system, such as cells of the innate or adaptive immunity, e.g.,myeloid or lymphoid cells, including lymphocytes, typically T-cellsand/or NK cells. Other exemplary cells include stem cells, such asmultipotent and pluripotent stem cells, including induced pluripotentstem cells (iPSCs). The cells typically are primary cells, such as thoseisolated directly from a subject and/or isolated from a subject andfrozen. In some embodiments, the cells include one or more subsets ofT-cells or other cell types, such as whole T-cell populations, CD4+cells, CD8+ cells, and subpopulations thereof, such as those defined byfunction, activation state, maturity, potential for differentiation,expansion, recirculation, localization, and/or persistence capacities,antigen-specificity, type of antigen receptor, presence in a particularorgan or compartment, marker or cytokine secretion profile, and/ordegree of differentiation. With reference to the subject to be treated,the cells may be allogeneic and/or autologous. Among the methods includeoff-the-shelf methods. In some aspects, such as for off-the-shelftechnologies, the cells are pluripotent and/or multipotent, such as stemcells, such as induced pluripotent stem cells (iPSCs). In someembodiments, the methods include isolating cells from the subject,preparing, processing, culturing, and/or engineering them, as describedherein, and re-introducing them into the same patient, before or aftercryopreservation.

Among the sub-types and subpopulations of T-cells and/or of CD4+ and/orof CD8+ T-cells are naive T (TN) cells, effector T-cells (TEFF), memoryT-cells and sub-types thereof, such as stem cell memory T (TSCM),central memory T (TCM), effector memory T (TEM), or terminallydifferentiated effector memory T-cells, tumor-infiltrating lymphocytes(TL), immature T-cells, mature T-cells, helper T-cells, cytotoxicT-cells, mucosa-associated invariant T (MALT) cells, naturally occurringand adaptive regulatory T (Treg) cells, helper T-cells, such as TH1cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells,follicular helper T-cells, alpha/beta T-cells, and delta/gamma T-cells.

In some embodiments, the cells are natural killer (NK) cells. In someembodiments, the cells are monocytes or granulocytes, e.g., myeloidcells, macrophages, neutrophils, dendritic cells, mast cells,eosinophils, and/or basophils.

The cells may be genetically modified to reduce expression or knock outendogenous TCRs. Such modifications are described in Mol Ther NucleicAcids. 2012 December: 1(12): e63; Blood. 2011 Aug. 11; 118(6):1495-503:Blood. 2012 Jun. 14; 119(24): 5697-5705; Torikai, Hiroki et al “HLA andTCR Knockout by Zinc Finger Nucleases: Toward “off-the-Shelf” AllogeneicT-Cell Therapy for CD19+ Malignancies.” Blood 116.21 (2010): 3766;Blood. 2018 Jan. 18; 131(3):311-322. doi: 10.1182/blood-2017-05-787598;and WO2016069283, which are incorporated by reference in their entirety.

The cells may be genetically modified to promote cytokine secretion.Such modifications are described in Hsu C, Hughes M S, Zheng Z, Bray RB, Rosenberg S A, Morgan R A. Primary human T lymphocytes engineeredwith a codon-optimized IL-15 gene resist cytokine withdrawal-inducedapoptosis and persist long-term in the absence of exogenous cytokine. JImmunol. 2005:175:7226-34; Quintarelli C, Vera J F, Savoldo B, GiordanoAttianese G M, Pule M, Foster A E, Co-expression of cytokine and suicidegenes to enhance the activity and safety of tumor-specific cytotoxic Tlymphocytes. Blood. 2007; 110:2793-802; and Hsu C, Jones S A, Cohen C J,Zheng Z, Kerstann K, Zhou J, Cytokine-independent growth and clonalexpansion of a primary human CD8+ T-cell clone following retroviraltransduction with the IL-15 gene. Blood. 2007; 109:5168-77.

Mismatching of chemokine receptors on T-cells and tumor-secretedchemokines has been shown to account for the suboptimal trafficking ofT-cells into the tumor microenvironment. To improve efficacy of therapy,the cells may be genetically modified to increase recognition ofchemokines in tumor micro environment. Examples of such modificationsare described in Moon, E K Carpenito, C Sun, J Wang, L C Kapoor, VPredina, J Expression of a functional CCR2 receptor enhances tumorlocalization and tumor eradication by retargeted human T-cellsexpressing a mesothelin-specific chimeric antibody receptor.Clin CancerRes. 2011; 17: 4719-4730; and Craddock, J ALu, A Bear, A Pule, MBrenner, M K Rooney, C M et al. Enhanced tumor trafficking of GD2chimeric antigen receptor T-cells by expression of the chemokinereceptor CCR2b. J Immunother. 2010; 33: 780-788.

The cells may be genetically modified to enhance expression ofcostimulatory/enhancing receptors, such as CD28 and 41BB.

Adverse effects of T-cell therapy can include cytokine release syndromeand prolonged B-cell depletion. Introduction of a suicide/safety switchin the recipient cells may improve the safety profile of a cell-basedtherapy. Accordingly, the cells may be genetically modified to include asuicide/safety switch. The suicide/safety switch may be a gene thatconfers sensitivity to an agent, e.g., a drug, upon the cell in whichthe gene is expressed, and which causes the cell to die when the cell iscontacted with or exposed to the agent. Exemplary suicide/safetyswitches are described in Protein Cell. 2017 August; 8(8): 573-589. Thesuicide/safety switch may be HSV-TK. The suicide/safety switch may becytosine daminase, purine nucleoside phosphorylase, or nitroreductase.The suicide/safety switch may be RapaCIDe™, described in U.S. PatentApplication Pub. No. US20170166877A1. The suicide/safety switch systemmay be CD20/Rituximab, described in Haematologica. 2009 September;94(9): 1316-1320. These references are incorporated by reference intheir entirety.

The TCR may be introduced into the recipient cell as a split receptorwhich assembles only in the presence of a heterodimerizing smallmolecule. Such systems are described in Science. 2015 Oct. 16:350(6258): aab4077, and in U.S. Pat. No. 9,587,020, which are herebyincorporated by reference.

In some embodiments, the cells include one or more nucleic acids, e.g.,a polynucleotide encoding a TCR disclosed herein, wherein thepolynucleotide is introduced via genetic engineering, and therebyexpress recombinant or genetically engineered TCRs as disclosed herein.In some embodiments, the nucleic acids are heterologous, i.e., normallynot present in a cell or sample obtained from the cell, such as oneobtained from another organism or cell, which for example, is notordinarily found in the cell being engineered and/or an organism fromwhich such cell is derived. In some embodiments, the nucleic acids arenot naturally occurring, such as a nucleic acid not found in nature,including one comprising chimeric combinations of nucleic acids encodingvarious domains from multiple different cell types.

The nucleic acids may include a codon-optimized nucleotide sequence.Without being bound to a particular theory or mechanism, it is believedthat codon optimization of the nucleotide sequence increases thetranslation efficiency of the mRNA transcripts. Codon optimization ofthe nucleotide sequence may involve substituting a native codon foranother codon that encodes the same amino acid, but can be translated bytRNA that is more readily available within a cell, thus increasingtranslation efficiency. Optimization of the nucleotide sequence may alsoreduce secondary mRNA structures that would interfere with translation,thus increasing translation efficiency.

A construct or vector may be used to introduce the TCR into therecipient cell. Exemplary constructs are described herein.Polynucleotides encoding the alpha and beta chains of the TCR may in asingle construct or in separate constructs. The polynucleotides encodingthe alpha and beta chains may be operably linked to a promoter, e.g., aheterologous promoter. The heterologous promoter may be a strongpromoter, e.g., EF1alpha, CMV, PGK1, Ubc, beta actin, CAG promoter, andthe like. The heterologous promoter may be a weak promoter. Theheterologous promoter may be an inducible promoter. Exemplary induciblepromoters include, but are not limited to TRE, NFAT, GAL4, LAC, and thelike. Other exemplary inducible expression systems are described in U.S.Pat. Nos. 5,514,578; 6,245,531; 7,091,038 and European Patent No.0517805, which are incorporated by reference in their entirety.

The construct for introducing the TCR into the recipient cell may alsocomprise a polynucleotide encoding a signal peptide (signal peptideelement). The signal peptide may promote surface trafficking of theintroduced TCR. Exemplary signal peptides include, but are not limitedto CD8 signal peptide, immunoglobulin signal peptides, where specificexamples include GM-CSF and IgG kappa. Such signal peptides aredescribed in Trends Biochem Sci. 2006 October:31(10):563-71. Epub 2006Aug. 21; and An, et al. “Construction of a New Anti-CD19 ChimericAntigen Receptor and the Anti-Leukemia Function Study of the TransducedT-cells.” Oncotarget 7.9 (2016): 10638-10649. PMC. Web. 16 Aug. 2018;which are hereby incorporated by reference.

In some cases, e.g., cases where the alpha and beta chains are expressedfrom a single construct or open reading frame, or cases wherein a markergene is included in the construct, the construct may comprise aribosomal skip sequence. The ribosomal skip sequence may be a 2Apeptide, e.g., a P2A or T2A peptide. Exemplary P2A and T2A peptides aredescribed in Scientific Reports volume 7, Article number: 2193 (2017),hereby incorporated by reference in its entirety. In some cases, aFURIN/PACE cleavage site is introduced upstream of the 2A element.FURIN/PACE cleavage sites are described in, e.g.,http://www.nuolan.net-substrates.html. The cleavage peptide may also bea factor Xa cleavage site. In cases where the alpha and beta chains areexpressed from a single construct or open reading frame, the constructmay comprise an internal ribosome entry site (IRES).

The construct may further comprise one or more marker genes. Exemplarymarker genes include but are not limited to GFP, luciferase, HA, lacZ.The marker may be a selectable marker, such as an antibiotic resistancemarker, a heavy metal resistance marker, or a biocide resistant marker,as is known to those of skill in the art. The marker may be acomplementation marker for use in an auxotrophic host. Exemplarycomplementation markers and auxotrophic hosts are described in Gene.2001 Jan. 24:263(1-2):159-69. Such markers may be expressed via an IRES,a frameshift sequence, a 2A peptide linker, a fusion with the TCR, orexpressed separately from a separate promoter.

Exemplary vectors or systems for introducing TCRs into recipient cellsinclude, but are not limited to Adeno-associated virus, Adenovirus,Adenovirus+Modified vaccinia, Ankara virus (MVA), Adenovirus+Retrovirus,Adenovirus+Sendai virus, Adenovirus+Vaccinia virus, Alphavirus (VEE)Replicon Vaccine, Antisense oligonucleotide, Bifidobacterium longum,CRISPR-Cas9, E. coli, Flavivirus, Gene gun, Herpesviruses, Herpessimplex virus. Lactococcus lactis, Electroporation, Lentivirus,Lipofection, Listeria monocytogenes, Measles virus, Modified VacciniaAnkara virus (MVA), mRNA Electroporation, Naked/Plasmid DNA,Naked/Plasmid DNA+Adenovirus, Naked/Plasmid DNA+Modified Vaccinia Ankaravirus (MVA), Naked/Plasmid DNA+RNA transfer, Naked/Plasmid DNA+Vacciniavirus, Naked/Plasmid DNA+Vesicular stomatitis virus, Newcastle diseasevirus, Non-viral, PiggyBac™ (PB) Transposon, nanoparticle-based systems,Poliovirus, Poxvirus, Poxvirus+Vaccinia virus, Retrovirus, RNA transfer,RNA transfer+NakedPlasmid DNA, RNA virus, Saccharomyces cerevisiae,Salmonella typhimurium, Semliki forest virus, Sendai virus. Shigelladysenteriae, Simian virus, siRNA, Sleeping Beauty transposon,Streptococcus mutans, Vaccinia virus, Venezuelan equine encephalitisvirus replicon, Vesicular stomatitis virus, and Vibrio cholera.

In preferred embodiments, the TCR is introduced into the recipient cellvia adeno associated virus (AAV), adenovirus, CRISPR-CAS9, herpesvirus,lentivirus, lipofection, mRNA electroporation, PiggyBac™ (PB)Transposon, retrovirus, RNA transfer, or Sleeping Beauty transposon.

In some embodiments, a vector for introducing a TCR into a recipientcell is a viral vector. Exemplary viral vectors include adenoviralvectors, adeno-associated viral (AAV) vectors, lentiviral vectors,herpes viral vectors, retroviral vectors, and the like. Such vectors aredescribed herein.

Exemplary embodiments of TCR constructs for introducing a TCR intorecipient cells is shown in FIG. 25. In some embodiments, a TCRconstruct includes, from the 5′-3′ direction, the followingpolynucleotide sequences: a promoter sequence, a signal peptidesequence, a TCR β variable (TCRβv) sequence, a TCR β constant (TCRβc)sequence, a cleavage peptide (e.g., P2A), a signal peptide sequence, aTCR α variable (TCRαv) sequence, and a TCR α constant (TCRαc) sequence.In some embodiments, the TCRβc and TCRαc sequences of the constructinclude one or more murine regions, e.g., full murine constant sequencesor human→murine amino acid exchanges as described herein. In someembodiments, the construct further includes, 3′ of the TCRαc sequence, acleavage peptide sequence (e.g., T2A) followed by a reporter gene. In anembodiment, the construct includes, from the 5′-3′ direction, thefollowing polynucleotide sequences: a promoter sequence, a signalpeptide sequence, a TCR β variable (TCRβv) sequence, a TCR α constant((TCRβc) sequence containing one or more murine regions, a cleavagepeptide (e.g., P2A), a signal peptide sequence, a TCR α variable (TCRαv)sequence, and a TCR α constant (TCRαc) sequence containing one or moremurine regions, a cleavage peptide (e.g., T2A), and a reporter gene.

FIG. 26 depicts an exemplary P526 construct backbone nucleotide sequencefor cloning TCRs into expression systems for therapy development.

FIG. 27 depicts an exemplary construct sequence for cloning patientneoantigen-specific TCR, clonotype I into expression systems for therapydevelopment.

FIG. 28 depicts an exemplary construct sequence for cloning patientneoantigen-specific TCR, clonotype 3 into expression systems for therapydevelopment.

Also provided are isolated nucleic acids encoding TCRs, vectorscomprising the nucleic acids, and host cells comprising the vectors andnucleic acids, as well as recombinant techniques for the production ofthe TCRs.

The nucleic acids may be recombinant. The recombinant nucleic acids maybe constructed outside living cells by joining natural or syntheticnucleic acid segments to nucleic acid molecules that can replicate in aliving cell or replication products thereof. For purposes herein, thereplication can be in vitro replication or in vivo replication.

For recombinant production of a TCR the nucleic acid(s) encoding it maybe isolated and inserted into a replicable vector for further cloning(i.e., amplification of the DNA) or expression. In some aspects, thenucleic acid may be produced by homologous recombination, for example asdescribed in U.S. Pat. No. 5,204,244, incorporated by reference in itsentirety.

Many different vectors are known in the art. The vector componentsgenerally include one or more of the following: a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence, for example asdescribed in U.S. Pat. No. 5,534,615, incorporated by reference in itsentirety.

Exemplary vectors or constructs suitable for expressing a TCR, antibody,or antigen binding fragment thereof, include, e.g., the pUC series(Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla,Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series(Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, PaloAlto, Calif.). Bacteriophage vectors, such as AGTIO, AGTI 1, AZapII(Stratagene), AEMBL4, and ANMl 149, are also suitable for expressing aTCR disclosed herein.

XVIII. Treatment Overview Flow Chart

FIG. 29 is a flow chart of a method for providing a customized,neoantigen-specific treatment to a patient, in accordance with anembodiment. In other embodiments, the method may include differentand/or additional steps than those shown in FIG. 29. Additionally, stepsof the method may be performed in different orders than the orderdescribed in conjunction with FIG. 29 in various embodiments.

The presentation models are trained 2901 using mass spectrometry data asdescribed above. A patient sample is obtained 2902. In some embodiments,the patient sample comprises a tumor biopsy and/or the patient'speripheral blood. The patient sample obtained in step 2902 is sequencedto identify data to input into the presentation models to predict thelikelihoods that tumor antigen peptides from the patient sample will bepresented. Presentation likelihoods of tumor antigen peptides from thepatient sample obtained in step 2902 are predicted 2903 using thetrained presentation models. Treatment neoantigens are identified 2904for the patient based on the predicted presentation likelihoods. Next,another patient sample is obtained 2905. The patient sample may comprisethe patient's peripheral blood, tumor-infiltrating lymphocytes (TIL),lymph, lymph node cells, and/or any other source of T-cells. The patientsample obtained in step 2905 is screened 2906 in vivo forneoantigen-specific T-cells.

At this point in the treatment process, the patient can either receiveT-cell therapy and/or a vaccine treatment. To receive a vaccinetreatment, the neoantigens to which the patient's T-cells are specificare identified 2914. Then, a vaccine including the identifiedneoantigens is created 2915. Finally, the vaccine is administered 2916to the patient.

To receive T-cell therapy, the neoantigen-specific T-cells undergoexpansion and/or new neoantigen-specific T-cells are geneticallyengineered. To expand the neoantigen-specific T-cells for use in T-celltherapy, the cells are simply expanded 2907 and infused 2908 into thepatient.

To genetically engineer new neoantigen-specific T-cells for T-celltherapy, the TCRs of the neoantigen-specific T-cells that wereidentified in vivo are sequenced 2909. Next, these TCR sequences arecloned 2910 into an expression vector. The expression vector 2910 isthen transfected 2911 into new T-cells. The transfected T-cells are 2912expanded. And finally, the expanded T-cells are infused 2913 into thepatient.

A patient may receive both T-cell therapy and vaccine therapy. In oneembodiment, the patient first receives vaccine therapy then receivesT-cell therapy. One advantage of this approach is that the vaccinetherapy may increase the number of tumor-specific T-cells and the numberof neoantigens recognized by detectable levels of T-cells.

In another embodiment, a patient may receive T-cell therapy followed byvaccine therapy, wherein the set of epitopes included in the vaccinecomprises one or more of the epitopes targeted by the T-cell therapy.One advantage of this approach is that administration of the vaccine maypromote expansion and persistence of the therapeutic T-cells.

XIX. Example Computer

FIG. 30 illustrates an example computer 3000 for implementing theentities shown in FIGS. 1 and 3. The computer 3000 includes at least oneprocessor 3002 coupled to a chipset 3004. The chipset 3004 includes amemory controller hub 3020 and an input/output (I/O) controller hub3022. A memory 3006 and a graphics adapter 3012 are coupled to thememory controller hub 3020, and a display 3018 is coupled to thegraphics adapter 3012. A storage device 3008, an input device 3014, andnetwork adapter 3016 are coupled to the I/O controller hub 3022. Otherembodiments of the computer 3000 have different architectures.

The storage device 3008 is a non-transitory computer-readable storagemedium such as a hard drive, compact disk read-only memory (CD-ROM),DVD, or a solid-state memory device. The memory 3006 holds instructionsand data used by the processor 3002. The input interface 3014 is atouch-screen interface, a mouse, track ball, or other type of pointingdevice, a keyboard, or some combination thereof, and is used to inputdata into the computer 3000. In some embodiments, the computer 3000 maybe configured to receive input (e.g., commands) from the input interface3014 via gestures from the user. The graphics adapter 3012 displaysimages and other information on the display 3018. The network adapter3016 couples the computer 3000 to one or more computer networks.

The computer 3000 is adapted to execute computer program modules forproviding functionality described herein. As used herein, the term“module” refers to computer program logic used to provide the specifiedfunctionality. Thus, a module can be implemented in hardware, firmware,and/or software. In one embodiment, program modules are stored on thestorage device 3008, loaded into the memory 3006, and executed by theprocessor 3002.

The types of computers 3000 used by the entities of FIG. 1 can varydepending upon the embodiment and the processing power required by theentity. For example, the presentation identification system 160 can runin a single computer 3000 or multiple computers 3000 communicating witheach other through a network such as in a server farm. The computers3000 can lack some of the components described above, such as graphicsadapters 3012, and displays 3018.

Supplementary Table 1 Predicted Ranks of Mutations with Pre-ExistingResponse MHCFlurry, MHCFlurry, MHCFlurry, Peptide MS Mutation ID PatientID TPM > 0 TPM > 1 TPM > 2 Model, TPM > 1 Full MS Model KARS_D356H 394281 44 36 26 5 NUP98_A359D 3942 13 8 7 0 0 CASP8_F67V 3971 13 3 2 3 1KRAS_G12D 3995 36 21 18 2 2 RNF213_N17025 3995 0 0 0 7 7 TUBGCP2_P293L3995 2 2 2 8 6 H3F3B_A48T 4007 33 23 21 13 0 SKIV2L_R653H 4007 2 1 1 1517 API5_R243Q 4032 52 31 27 10 1 PHLPP1_G566E 4032 54 33 29 72 67RNF10_E572K 4032 43 23 22 46 46 ZFYVE27_R6H 4069 35 23 22 0 0CADPS2_R1266H 4136 23 22 22 4 5 KIAA0368_5186F 4136 2 2 2 1 0FLNA_R2049C NCI-3784 91 85 81 31 5 KIF16B_L1009P NCI-3784 22 21 19 74 69SON_R1927C NCI-3784 37 35 32 105 83 KIF1BP_P2465 NCI-3903 66 35 32 22 7MAGEA6_E168K NCI-3998 15 10 9 1 0 MED13_P1691S NCI-3998 5 3 2 0 1PDS5A_Y1000F NCI-3998 13 8 7 6 4 CDK4_R71L patient1 56 23 20 5 0DNAH17_H8302Y patient1 42 80 59 112 77 GCN1_L2330P patient1 59 25 22 3 1BRWD1_R925W patient2 80 62 58 74 75 PARG_Y427N patient2 88 69 65 51 49Median 35.5 23 21.5 9 5

Supplementary Table 2 Demographics of NSCLS Patients Year of InitialSystemic Current Age Range (Lung Cancer) Tumor Stage (At Location ofNSCLC-Directed Anti-PD(L)-1 Patient ID (Years) Gender Race DiagnosisEnrollment) Primary Tumor Histological Type Therapy Therapy HLA-A1-001-002 81-90 Male White 2010 IIIB Lung Non-squamous CarboplantinNivolumab A*01:01 1-024-001 81-90 Male White 2016 IV Lung SarcomatoidPembrolizumab A*32:01 pulmonary carcinoma 1-024-002 51-60 Female White2016 IV Lung Adenocarcinoma CARBOplatin, Nivolumab A*68:01 DOCEtaxel,Bevacizumab, Ramucirumab, Pemetrexed Disodium 1-038-001 61-70 Male White2016 IV Lung Adenocarcinoma premetexed, Cisplatin Nivolumab A*69:011-050-001 71-80 Female White 2015 IIIB Lung Adenocarcinoma ETOPOSIDE,cisplatin Nivolumab A*29:02 CU05 71-80 Female White 2013 IV Lung LungSquamous carboplatin plus Nivolumab A*24:02 pemetrexed CU04 61-70 FemaleHispanic 2013 I Lung Adenocarcinoma durvalumab plus A*24:26 or Latinotremelimumab CU03 61-70 Male Black or 2016 I Lung Lung Squamous n/aA*23:01 African American CU02 61-70 Male White 2016 I Lung Lung Squamouscarboplatin + gemcitabine n/a A*02:01 Non- Normal DNA Tumor DNA RNA PFExpressed synonymous Median Exon Median Exon Unique Reads Median HLA-AHLA-B HLA-B HLA-C HLA-C Mutations Mutations Coverage Coverage (M) KnownDrivers Likely Drivers VAF A*01:01 B*08:01 B*51:01 C*01:02 C*07:01 122232 145 552 173 KRAS_G12D, STK11_G52fs 0.22 TP53_R213* A*03:01 B*27:05B*27:05 C*02:02 C*02:02  83 143 165 508 131.9 KRAS_G12C, PML_E43*, 0.093TP53_R280T NF2_R341* A*68:01 B*40:02 B*40:27 C*03:04 C*03:04  38  69 190454 114.4 KRAS_G12S, STK11_E199* 0.182 TP53_Q331* A*01:02 B*41:01B*49:01 C*17:01 C*07:01 158 265 158 983 311.8 KRAS_G12V KDMSC_E303* 0.19A*26:01 B*44:03 B*07:05 C*16:01 C*15:05  53  92 117 556 119 0.059A*68:02 B*14:02 B*15:17 C*07:01 C*08:02  65 109 191 448  83.6 0.095A*26:01 B*18:01 B*38:01 C*12:03 C*12:03 336 511 213 552 240.4 TP53_R158GNFKBIE_G41fs, 0.224 CDH1_Q346*, NF1_D2163fs, MED12_R730* A*01:01 B*08:01B*15:03 C*01:02 C*12:03 105 187 114 830 182.1 0.242 A*03:01 B*07:02B*57:01 C*07:02 C*06:02 102 174 105 738 185.3 TP53_R175H ATR_Q195* 0.32

Supplementary Table 3Peptides Tested for T-Cell Recognition In NSCLC Patients Indi- Most vid-Prob- ual Indi- able Pep- vid- Re- Most tide ual Pool stric- ProbableRe- Pep- Re- tion Re- sponse tide sponse covered Full striction SEQ (AnyRe- (Any Muta- Pro- by Full MS covered Pa- Pep- ID Time sponse Time tiontein MS Model MHCFlurry MHCFlurry by tient tide NO: Point) Notes Pool IDPoint) Mutation Type Gene Effect TPM Model Rank Rank (nM) MHCFlurry 1-HSPF  52 N 1-001-002_pool_1 N chr15_28215653_C_A snp HERC2 A2060s  41.9HLA-  0  95  5169.68205 FALSE 001- TATSL C*01:02 002 1-0 DPEE  53 N1-001-002_pool_1 N chr17_59680958_C_T snp CLTC S989L 272.1 HLA-  1  61 3455.25069 TRUE 01- VLVTV B*51:01 002 1- ELDP  54 N 1-001-002_pool_1 Nchr13_30210371_C_A snp KATNAL1 D407Y  12.81 HLA-  2   1    24.2177849TRUE 001- DIQL A*01:01 002 EY 1- TPLT  55 N 1-001-002_pool_1 Nchr5_78100974_A_T snp AP3B1 S817T  44.4 HLA-  3   2    48.9740194 TRUE001- DVTL 002 B*08:01 1- DGVG  56 N 1-001-002_pool_1 Nchr12_25245350_C_T snp KRAS G12D  40.75 HLA-  4  89  4714.29522 TRUE001- KSAL 002 B*08:01 1- YITV  57 N 1-001-002_pool_1 Nchr17_28339664_G_T snp TNFAIP1 R48L  45.62 HLA-  5  26   973.417701 TRUE001- RALT 002 L B*08:01 1- TPSA  58 N 1-001-002_pool_1 Nchr15_81319417_T_C snp STARD5 M108V   1.95 HLA-  6  39  2031.48603 TRUE001- AVKL 002 I B*51:01 1- WPVL  59 N 1-001-002_pool_1 Nchr3_179025167_AAC_A del_fs ZMAT3 V240fs  14.99 HLA-  7  16   600.564752TRUE 001- LLNV B*51:01 002 1- ELNA  60 N 1-001-002_pool_1 Nchr18_79943341_G_A snp PQLC1 R109C  33.89 HLA-  8   5    62.0439997 TRUE001- RRCS B*08:01 002 F 1- QMKN  61 N 1-001-002_pool_1 Nchr9_127663287_G_T snp STXBP1 R171L  38.76 HLA-  9  20   674.64733 TRUE001- PILE B*08:01 002 L 1- LTEK  62 N 1-001-002_pool_2 NChr9_92719180_C_T snp BICD2 E489K  42.66 HLA- 10  10   428.744925 TRUE001- VSLL A*01:01 002 K 1- SPFT  63 N 1-001-002_pool_2 Nchr15_28215653_C_A snp HERC2 A2060S  41.9 HLA- 11   4    59.1155419 TRUE001- ATSL B*08:01 002 1- NVDM  64 N 1-001-002_pool_2 Nchr9_121353262_T_A snp STOM K93N 360.6 HLA- 12  30  1490.72261 TRUE 001-RTIS B*08:01 002 F 1- TSIV  65 N 1-001-002_pool_2 N chr4_39205691_C_Tsnp WDR19 A282V  18.12 HLA- 13 176  9862.33009 TRUE 001- VSQT B*08:01002 L 1- HIKI  66 N 1-001-002_pool_2 N chr13_73062087_C_T snp KLF5 T163I 25.77 HLA- 14  27  1122.27455 TRUE 001- EPVA B*08:01 002 I 1- DSPD  67N 1-001-002_pool_2 N chr20_44197575_C_T snp OSER1 S119N  20.7 HLA- 15471 21598.414 FALSE 001- GSNG C*01:02 002 L 1- YTAV  68 N1-001-002_pool_2 N chr12_562.48788_C_A snp ANKRD52 A559S  18.32 HLA- 16  0    11.5906737 TRUE 001- HYAA A*01:01 002 SY 1- VGAD  69 N1-001-002_pool_2 N chr12_25245350_C_T snp KRAS G12D  40.75 HLA- 17 37017985.3612 FALSE 001- GVGK C*01:02 002 SAL 1- MMPP  70 N1-001-002_pool_2 N chr17_32369404_A_T snp ZNF207 Q409L 186 HLA- 18 136 7609.76602 TRUE 001- LPGI B*51:01 002 1- FPYP  71 N 1-001-002_pool_2 Nchr5_109186272_G_T snp FER C759F  67.36 HLA- 19  38  1999.07208 TRUE001- GMTN B*51:01 002 Q 1- VTNH  72 N 1-024-001_pool_ 1 Ychr3_125552370_C_A snp OSBPL11 G489W  24.12 HLA-  0   7    77.009026TRUE 024- APLS A*32:01 001 W 1- GTKK  27 Y 1-024-001_pool_1 Ychr20_56513366_G_A snp RTFDC1 E177K  61.32 HLA-  1  70  2168.51668 TRUE024- DVDV A*03:01 001 LK 1- GLNV  34 N 1-024-001_pool_1 Ychr4_88390868_G_T snp HERC6 R218L   8.7 HLA-  2   4    59.675168 TRUE024- PVCQ A*03:01 001 SNK 1- VVVG  73 N 1-024-001 pool_1 Ychr12_25245351_C_A snp KRAS G12C  40.05 HLA-  3  11   133.648023 TRUE024- ACGV A*03:01 001 GK 1- AQFA  74 N 1-024-001_pool_1 Ychr9_89045819_C_A snp SHC3 E376D   8.88 HLA-  4  91  3715.42819 TRUE024- GKDQ A*32:01 001 TY 1- KVVL  75 N 1-024-001 pool 1 Ychr3_48591778_G_T snp COL7A1 R468S  25.42 HLA-  6  85  3234.15772 TRUE024- PSDV A*32:01 001 TSY 1- MLMK  76 N 1-024-001_pool_1 Ychr12_6959976_G_A snp PTPN6 E471K 105.4 HLA-  7   0    12.2301919 TRUE024- NIST A*03:01 001 K 1- DLAG  77 N 1-024-001_pool_1 Ychr11_123059991_C_G snp HSPA8 G201A 735.6 HLA-  9 353 18290.7955 TRUE024- GTFD B*27:05 001 V 1- LIFD  78 N 1-024-001_pool_1 Ychr11_123059991_C_G snp HSPA8 G201A 736.6 HLA- 11  57  1716.74204 FALSE024- LAGG C*02:02 001 TF 1- NVLI  79 N 1-024-001_pool_1 Ychr11_123059991_C_G snp HSPA8 G201A 736.5 HLA- 17 621 27984.1357 TRUE024- FDLA A*32:01 001 1- VVGA  80 N 1-024-001_pool_2 Nchr12_25245351_C_A snp KRAS G12C  40.05 HLA-  5  19   197.846108 TRUE024- CGVG A*03:01 001 K 1- VIML  81 N 1-024-001_pool_2 Nchr20_56513366_G_A snp ETFDC1 E177K  61.32 HLA-  8  10   122.750322 TRUE024- NGTK A*03:01 001 K 1- LAGG  82 N 1-024-001_pool_2 Nchr11_123059991_C_G snp HSPA8 G201A 736.6 HLA- 10 632 28384.8834 FALSE024- TFDV C*02:02 001 1- LRNS  83 N 1-024-001_pool_2 Nchr14_80906012_TC_T del_fs CEP128 R102fs  11.31 HLA- 12  46  1020.95087TRUE 024- GGEV B*27:05 001 F 1- VVLP  84 N 1-024-001_pool_2 Nchr3_48591778_G_T snp COL7A1 R468S  25.42 HLA- 13  62  1925.29397 TRUE024- SDVT A*32:01 001 SY 1- IFDL  85 N 1-024-001_pool_2 Nchr11_123059991_C_G snp HSPA8 G201A 736.6 HLA- 14 427 21255.2074 FALSE024- AGGT C*02:02 001 F 1- GLLD  86 N 1-024-001_pool_2 Nchr19_57575861_G_T snp ZNF416 Q49K  11.89 HLA- 15  24   354.82068 TRUE024- EAKR A*03:01 001 LLY 1- SVLL  87 N 1-024-001_pool_2 Nchr11_122789248_G_T snp UBASH3B G307V  12.11 HLA- 16  23   228.127132TRUE 024- PENY A*03:01 001 ITK 1- DLAG  88 N 1-024-001_pool_2 Nchr11_123059991_C_G snp HSPA8 G201A 736.6 HLA- 18 487 23357.3292 TRUE024- GTFD A*32:01 001 VS 1- IFDL  89 N 1-024-001_pool_2 Nchr11_123059991_C_G snp HSPA8 G201A 736.6 HLA- 19 563 25887.4267 FALSE024- AGGT C*02:02 001 FDV 1- AEWR  90 N 1-024-002_pool_1 Ychr3_122703943_C_G snp PARP14 P1095A 129.5 HLA-  0   8   126.397714 TRUE024- NGST A*68:01 002 SSL 1- YVSE  35 N 1-024-002_pool_1 Ychr2_43889858_G_A snp LRPPRC T1335I  79.08 HLA-  1   9   136.482378 TRUE024- KDVI A*68:01 002 SAK 1- EGSL  91 N 1-024-002_pool_1 Ychr18_62157782_C_A snp PIGN W83L  20.74 HLA-  2   6    88.2623459 TRUE024- GISH A*68:01 002 TR 1- IPAS  92 N 1-024-002_pool_1 Ychr13_109784018_C_A snp IR52 S679I  63.55 HLA-  3  16   224.278982 TRUE024- VSAP A*68:01 002 K 1- QDVS  24 Y 1-024-002_pool_1 Ychr9_64411223_T_G snp ANKRD20A4 M646R   8.92 HLA-  4  14   193.974327TRUE 024- VQVE A*68:01 002 R 1- LVVV  93 N 1-024-002_pool_1 Ychr12_25245351_C_T snp KRAS G12S  72.77 HLA-  6  41  1238.56407 TRUE024- GASG A*68:01 002 VGK 1- RATI  36 N 1-024-002_pool_1 Ychr7_1314632S3_A_T snp MKLN1 D521V  84.08 HLA-  7 266 16010.7063 FALSE024- VPEL C*03:04 002 1- SSAA  21 Y 1-024-002_pool_1 Y chr6_13711102_T_Asnp RANBP9 H135L  43.5 HLA-  8 103  4565.97417 FALSE 024- APFP C*03:04002 L 1- GVSK  94 N 1-024-002_pool_1 Y chr4_10116175_C_T snp WDR1 D26N134.5 HLA-  9 125  6797.60699 TRUE 024- IIGG A*68:01 002 NPK 1- EQNF  33not 1-024-002_pool_1 Y chr3_25791346_A_C snp OXSM K109T  12.82 HLA- 17156  9099.70986 TRUE 024- VSTS tested A*68:01 002 DIK indi- vid- ually1- RTQD  95 N 1-024-002_pool_2 Y chr9_64411223_T_G snp ANERD20A4 M646R  8.92 HLA-  5  53  1847.42359 TRUE 024- VSVQ A*68:01 002 VER 1- EAGN 96 N 1-024-002_pool_2 Y chr2_74046630_G_T snp TET3 G238V  56.35 HLA- 10 13   161.242762 TRUE 024- NSRV A*68:01 002 PR 1- RYVL  97 N1-024-002_pool_2 Y chr3_122703943_C_G snp PARP14 P1095A 129.5 HLA- 11176 10453.627 TRUE 024- HVVA A*68:01 002 A 1- VSKI  98 N1-024-002_pool_2 Y chr4_10116175_C_T snp WDR1 D26N 134.5 HLA- 12  38  954.724495 TRUE 024- IGGN A*68:01 002 PK 1- QPSG  99 N1-024-002_pool_2 Y chr12_14478436_GG_TT mnp ATF7IP G1021L 123.2 HLA- 13139  7795.97025 TRUE 024- VPTS A*68:01 002 L 1- DVSV 100 N1-024-002_pool_2 Y chr9_64411223_T_G snp ANKRD20A4 M6468   8.92 HLA- 14  7   123.489687 TRUE 024- QVER A*68:01 002 1- FVST  22 Y1-024-002_pool_2 Y chr3_25791346_A_C snp OXSM K109T  12.82 HLA- 15 128 7025.56581 FALSE 024- SDIK C*03:04 002 SM 1- FPVV  39 N1-024-002_pool_2 Y chr1_116062776_G_C snp SLC22A15 A396P   8.57 HLA- 16155  9082.40652 FALSE 024- NSHS C*03:04 002 L 1- APFP 101 N1-024-002_pool_2 Y chr6_13711102_T_A snp RAN8P9 H135L  43.5 HLA- 18 19611590.601 TRUE 024- LGDS A*68:01 002 AL 1- ATIV 102 N 1-024-002_pool_2 Ychr7_131463253_A_T snp MKLN1 D521V  84.08 HLA- 19 365 19785.1419 TRUE024- PELN A*68:01 002 EI 1- QEFA 103 N see 1-038-001_pool_1 Ychr2_219501883_G_T snp GMPPA G92V  21.6 HLA-  0  31  3481.07375 FALSE038- PLGT pool B*49:01 001 V re- sults 1- MNQV 104 not see1-038-001_pool_1 Y chr14_100354547_C_G snp WARS D148H 757.2 HLA- 12 42227180.1513 FALSE 038- LHAY tested pool C*07:01 001 indi- re- vid- sultsually 1- HEDV 105 not see 1-038-001_pool_1 Y chr8_96231911_C_G snp UQCRBD41H 174.8 HLA- 16 300 24830.2411 FALSE 038- KEAI tested pool B*49:01001 indi- re- vid- sults ually 1- GPYP 106 not see 1-038-001_pool_1 Ychr1_111242326_C_T snp CHI3L2 L379F 122.3 HLA-  1  19  1176.97782 FALSE038- FVQA tested pool B*49:01 001 V indi- re- vid- sults ually 1- YEHE107 not see 1-038-001_pool_1 Y chr8_96231911_C_G snp UQCRB D41H 174.8HLA-  2 212 22559.0306 FALSE 038- DVKE tested pool B*49:01 001 AI indi-re- vid- sults ually 1- EESV 108 not see 1-038-001_pool_1 Ychr1_15583354_CC_AG mnp AGMAT G105L   1.03 HLA-  3 109 17185.8013 FALSE038- MLLT tested pool B*49:01 001 V indi- re- vid- sults ually 1- IEED109 not see 1-038-001_pool_1 Y chr6_84215849_C_A snp CEP162 E82D  15.62HLA-  4 171 20568.515 FALSE 038- SAEK tested pool B*49:01 001 I indi-re- vid- sults ually 1- TEED 110 not see 1-038-001_pool_1 Ychr7_93105459_C_A snp SAMD9 M213I  68.23 HLA-  5 226 22894.2742 FALSE038- VKIK tested pool B*49:01 001 F indi- re- vid- sults ually 1- NEQS111 not see 1-038-001_pool_1 Y chrX_70375298_C_G snp KIF4A L625V  19.51HLA-  6 141 19054.8385 FALSE 038- KLLK tested pool B*49:01 001 V indi-re- vid- sults ually 1- VDNI 112 not see 1-038-001_pool_1 Ychr20_2654879_G_T snp NOP56 M167I  89.39 HLA-  7 119 17928.6022 FALSE038- IIQS tested pool B*49:01 001 I indi- re- vid- sults ually 1- YEHE 20 Y 1-038-001_pool_2 Y chr8_96231911_C_G snp UQCRB D41H 174.8 HLA-  9250 23419.567 FALSE 038- DVKE B*49:01 001 A 1- YVSE 113 not1-038-001_pool_2 Y chr17_2330604_G_A snp TSR1 H561Y  48.21 HLA- 10   0    6.07874308 FALSE 038- VPVS tested C*17:01 001 V indi- vid- ually 1-SELT 114 not 1-038-001_pool_2 Y chr19_37564705_G_C snp ZNF571 L575V 19.07 HLA- 11 159 19886.0407 FALSE 038- VHQR tested B*49:01 001 I indi-vid- ually 1- VGVG 115 not 1-038-001_pool_2 Y chr12_25245350_C_A snpKRAS G12V  91.89 HLA- 13 388 26432.7668 FALSE 038- GKSA tested C*17.01001 L indi- vid- ually 1- DMNQ 116 not 1-038-001_pool_2 Ychr14_100354547_C_G snp WARS D148H 757.2 HLA- 14  64 10286.4383 FALSE038- VLHA tested C*07:01 001 Y indi- vid- ually 1- NEKG 117 not1-038-001_pool_2 Y chr17_512940400_G_T snp UTP18 M547I  63.21 HLA- 15339 25564.2874 FALSE 038- KALI tested C*07:01 001 Y indi- vid- ually 1-TEYK 118 not 1-038-001_pool_2 Y chr12_25245350_C_A snp KRAS G12V  91.89HLA- 17 233 23113.572 FALSE 038- LVVV tested B*49:01 001 GAV indi- vid-ually 1- QEFA 119 not 1-038-001_pool_2 Y chr2_219501883_G_T snp GMPPAG92V  21.6 HLA- 18 338 25558.5468 FALSE 038- PLGT tested B*49:01 001 VGindi- vid- ually 1- QEVR 120 not 1-038-001_pool_2 Y chr17_4085728_C_Asnp ZZEF1 G863V  63 HLA- 19 124 18359.7482 FALSE 038- NTLL testedB*49-01 001 NV indi- vid- ually 1- VEML 121 not 1-038-001_pool_2 Ychr4_168427109_C_A snp DDX60L A631S  44.71 HLA-  8 267 23949.2394 FALSE038- GLIS tested B*49:01 001 C indi- vid- ually 1- LFHD 122 N1-050-001_pool_1 N chr1_193097666_T_C snp GLRX2 N94S  17.92 HLA-  0   1   44.54051 TRUE 050- MNVS A*29:02 001 Y 1- ISTF 123 not1-050-001_pool_1 N chr17_80346815_G_T snp RNF213 R2827L 330.6 HLA- 10322 22721.4424 FALSE 050- RQCA tested C*16:01 001 L indi- vid- ually 1-YNTD 124 not 1-050-001_pool_1 N chr15_26580447_G_T snp GABRB3 T185N  2.2 HLA- 16  20   447.152559 TRUE 050- DIEF tested A*29:02 001 Y indi-vid- ually 1- EETP 125 N 1-050-001_pool_1 N chr21_31266125_T_A snp TIAM1Y283F  13.99 HLA-  1  26   537.02592 TRUE 050- PFSN B*44:03 001 Y 1-QASG 126 not 1-050-001_pool_1 N chr22_30893501_T_C snp OSBP2 Y677H  7.86 HLA- 19 109  7506.81856 TRUE 050- NHHV tested B*44:03 001 W indi-vid- ually 1- EEVT 127 not 1-050-001_pool_1 N chr18_5419733_G_A snpEPB41L3 S49SL  51.69 HLA-  2  17   390.306194 TRUE 050- PILA testedB*44:03 001 I indi- vid- ually 1- IEHN 128 not 1-050-001_pool_1 Nchr3_52617347_T_G snp PBRM1 D578A  65.64 HLA-  3  10   186.953378 TRUE050- IRNA tested B*44:03 001 KY indi- vid- ually 1- AERL 129 not1-050-001_pool_1 N chr14_103339252_G_T snp EIF5 M275I  89.97 HLA-  5  34 1075.19965 TRUE 050- DVKA tested B*44:03 001 I indi- vid- ually 1- LFQQ130 not 1-050-001_pool_1 N chr17_80346815_G_T snp RNF213 R2827L 330.6HLA-  6  54  2855.46701 TRUE 050- GKDL tested A*29:02 001 QQY indi- vid-ually 1- DTSP 131 not 1-050-001_pool_1 N chr5_73074790_T_C snp FCHO2L543S  43.6 HLA-  8  91  5750.39585 TRUE 050- VAVA tested A*26:01 001 Lindi- vid- ually 1- AEET 132 N 1-050-001_pool_2 N chr21_31266125_T_A snpTIAm1 Y283F  13.99 HLA-  9  16   364.187996 TRUE 050- PPFS B*44:03 001NY 1- AAKA 133 not 1-050-001_pool_2 N chr3_47661451_C_G snp SMARCC1E721D  39.53 HLA- 11 307 22125.437 FALSE 050- ALED tested C*16:01 001 Findi- vid- ually 1- EVTP 134 not 1-050-001_pool_2 N chr18_5419733_G_Asnp ERB41L3 S495L  51.69 HLA- 12 125  9269.11767 TRUE 050- ILAI testedA*26:01 001 R indi- vid- ually 1- DVKA 135 not 1-050-001 pool _2 Nchr14_103339252_G_T snp EIF5 M275I  89.97 HLA- 13  90  5692.75283 TRUE050- IGPL tested A*26:01 001 V indi- vid- ually 1- NETP 136 not1-050-001_pool_2 N chr7_79453094_C_A snp MAGI2 G76V   2.29 HLA- 14  13  253.431553 TRUE 050- VAVL tested B*44:03 001 TI indi- vid- ually 1-LFVV 137 not 1-050-001_pool_2 N chr1_159535913_A_T snp OR10I5 L32Q   0.9HLA- 15   9   139.510048 TRUE 050- FQTV tested A*29:02 001 Y indi- vid-ually 1- AEAE 138 not 1-050-001_pool_2 N chr14_103339252_G_T snp EIF5M275I  89.97 HLA- 17  38  1465.22509 TRUE 050- RLDV tested B*44:03 001KAI indi- vid- ually 1- ASGN 139 not 1-050-001_pool_2 Nchr22_30893501_T_C snp OSBP2 Y677H   7.86 HLA- 18 173 13216.9384 FALSE050- HHVW tested C*16:01 001 indi- vid- ually 1- KLFH 140 not1-050-001_pool_2 N chr21_193097666_T_C snp GLRX2 N945  17.92 HLA-  4  21  453.621334 TRUE 050- DMNV tested A*29:02 001 SY indi- vid- ually 1-ETPP 141 not 1-050-001_pool_2 N chr21_31266125_T_A snp TIAM1 Y283F 13.99 HLA-  7 172 13162.6216 TRUE 050- FSNY tested A*26:01 001 NTLindi- vid- ually CU04 DENI  23 Y CU04_pool_1 Y chr4_22413213_C_A snpADGRA3 C734F  20.67 HLA-  0   2     8.27203164 TRUE TTIQ B*18:01 F CU04MELK 142 N CU04_pool_1 Y chr1_37874128_G_C snp INPP5B Q606E  36.85 HLA- 1   5    13.0510076 TRUE VESF B*18:01 CU04 EHIP 143 N CU04_ pool_1 Ychr3_9943508_G_C snp CRELD1 Q347H  29.9 HLA-  2 303  4218.0095 TRUE ESAGB*38:01 F CU04 YHGD 144 N CU04_pool_1 Y chr12_7066530_C_T snp C1S P295L157.5 HLA-  3  12    76.7416543 TRUE PMPC B*38:01 L CU04 DEER 145 NCU04_pool_1 Y chr7_5752914_T_C snp RNF216 M45V  49.2 HLA-  4  29  387.328968 TRUE IPVL B*18:01 CU04 EVAD  25 Y CU04_pool_1 Ychr1_52268541_A_C snp ZFYVE9 K845T  70.08 HLA-  5   7    38.7340629 TRUEAATL A*26:01 TM CU04 IEVE 146 N CU04_pool_1 y chr7_135598004_C_G snpNUP205 L691V  42.37 HLA-  6  21   209.301169 TRUE VNEI B*18:01 CU04 DTVE 26 Y  CU04_pool_1 Y chr14_34713369_C_A snp CFL2 D66Y  16.65 HLA-  7   9   42.7267485 TRUE YPYT A*26:01 SF CU04 VEIE 147 N CU04_pool_1 Ychr11_62827178_C_G snp STX5 E134Q  83.43 HEA-  8   3    11.6727539 TRUEQLTY B*18:01 CU04 LELK 148 N CU04_pool_1 Y chr7_138762364_G_T snpATP6V0A4 P163H  47.21 HLA-  9   0     3.63590379 TRUE AVHA B*18:01 YCU04 EEAD 149 N CU04_pool_2 N chr6_10556704_C_T snp GCNT2 P94L  25.19HLA- 10   1     6.48490966 TRUE FLLA B*18:01 Y CU04 ENIT 150 NCU04_pool_2 N chr4_22413213_C_A snp ADGRA3 C734F  20.67 HLA- 11  16  135.44155 TRUE TIQF A*26:01 Y CU04 FHAT 151 N CU04_pool_2 Nchr14_75117203_C_G snp NEK9 D252H  20.29 HLA- 12   8    39.1165673 TRUENPLN B*38:01 L CU04 VFKD 152 N CU04_pool_2 N chrX_40597563_G_A snpATP6AP2 E145K  88.26 HLA- 13  45  1080.8332 TRUE LSVT B*38:01 L CU04QAVA 153 N CU04_pool_2 N chr17_42104792_T_A snp DHX58 M513L  35.87 HLA-14 136  6872.44 TRUE AVQK C*12:03 L CU04 IQDQ 154 N CU04_pool_2 Nchr2_67404159_G_C snp ETAA1 E493Q  38.47 HLA- 15  59  1665.0162 TRUEIQNC B*38:01 I CU04 VAKG 155 N CU04_pool_2 N chr2_85395579_C_T snp CAPGE314K 151.7 HLA- 16 107  5236.61406 TRUE FISR C*12:03 M CU04 QTKP 156 NCU04_pool_2 N chr2_32487684_AG_A del_fs BIRC6 G2619fs 111.7 HLA- 17  47 1143.73481 TRUE ASLL A*26:01 Y CU04 DHFE 157 N CU04_pool_2 Nchr1_220024376_C_G snp EPR5 M277I  76.64 HLA- 18   6    29.8996386 TRUETIIK B*18:01 Y CU04 VEYP 158 N CU04_pool_2 N chr14_34713369_C_A snp CFL2D66Y  16.65 HLA- 19   4    12.3783994 TRUE YTSF B*18:01 CU05 SVSD 159 NCU05_pool_1 N chr12_15670870_G_C snp EPS8 Q64E  52.56 HLA-  0   1    6.0399624 TRUE ISEY A*68:02 RV CU05 YTFE 160 N CU05_pool_1 Nchr1_22865138C_G snp EPH82 A410G  74.99 HLA-  1  22   132.877429 TRUEIQGV A*68:02 NGV CU05 IYTS 161 N CU05_pool_1 N chr10_73293336_T_C snpCFAP70 E636G  30.45 HLA-  2  17    46.3526841 TRUE SGQL A*24:02 QLF CU05FATP 162 N CU05_pool_1 N chr17_80345147_A_T snp RNF213 D2271V 735.3 HLA- 4  16    43.8761927 TRUE SLHT A*68:02 SV CU05 AVSK 163 N CU05_pool_1 Nchr14_77026556_T_A snp IRF2BPL M413L  58.51 HLA-  5 274 13566.6012 TRUEPGLD A*68:02 YEL CU05 KYIN 164 N CUO5_pool_1 N chr19_2328426_C_T snpLSM7 D20N  76.01 HLA-  8  32   318.671051 TRUE KTIR A*24:02 V CU05 ETTE165 N CU05_pool_1 N chr6_80040624_G_A snp TTK G804E  17.14 HLA-  9  37  398.324158 TRUE EMKY A*68:02 VL CU05 VVSH 166 N CU05_pool_l Nchr4_106232956_C_G snp TBCK D478H  71.17 HLA- 11 235 10875.8686 TRUEPHLV A*68:02 YW CU05 DIFQ 167 N CU05_pool_1 N chr1_198754369_C_A snpPTPRC L1204I 104.6 HLA- 13  36   394.198029 TRUE VVKA A*68:02 I CUO5FAFD 168 N CU05_pool_l N chr14_77026556_T_A snp IRF2BPL M413L  58.51HLA- 18  65  1067.11951 TRUE AVSK A*68:02 PGL CU05 SVSD 169 NCU05_pool_2 N chr12_15670870_G_C snp EP58 Q64E  52.56 HLA-  3  94 2050.45825 TRUE I5EY A*68:02 R CU05 YTFE 170 N CU05_pool_2 Nchr1_22865138_C_G snp EPH82 A410G  74.99 HLA-  6  11    26.6362167 TRUEIQGV A*68:02 CU05 ATPS 171 N CU05_pool_2 N chr17_80345147_A_T snp RNF213D2271V 735.3 HLA-  7  25   177.027506 TRUE LHTS A*68:02 V CU05 DFAT 172N CU05_pool_2 N chr17_80345147_A_T snp RNF213 D2271V 735.3 HLA- 10 185 7619.02631 TRUE PSLH A*68:02 TSV CU05 KYIN 173 N CU05_pool_2 Nchr19_2328426_C_T snp LSM7 D20N  76.01 HLA- 12  42   538.209517 TRUEKTIR A*24:02 VKF CU05 SVKP 174 N CU05_pool_2 N chr17_35363437_C_T snpSLFN11 R124H  91.5 HLA- 14  88  1897.58723 TRUE HLCS A*68:02 L CU05 DISE175 N CU05_pool_2 N chr12_15670870_G_C snp EP58 Q64E  52.56 HLA- 15  59  885.161001 TRUE YRVEF A*68:02 HL CU05 WVVS 176 N CU05_pool_2 Nchr4_106232956_C_G snp TBCK D478H  71.17 HLA- 16  15    40.725305 TRUEHPHL A*68:02 V CU05 KVFK 177 N CU05_pcol_2 N chrX_24810777_G_A snp POLA1E1017K  19.31 HLA- 17  61   954.869111 TRUE LGNK A*68:02 V CU05 VSKP 178N CU05_pool_2 N chr14_77026556_T_A snp IRF2BPL M413L  58.1 HLA- 19 25812457.5646 TRUE GLDY A*68:02 EL CU02 SPSK  37 not see CU02_pool_1 Ychr12_132750694_G_T snp ANKLE2 P266T  43.78 HLA-  0   7    20.5140939TRUE TSLT tested pool B*07:02 L indi- re- vid- sults ually CU02 ASAD  40not see CU02_pool_1 Y chr16_1977246_A_G snp TBL3 I545V  26.23 HLA-  1 20    77.5504026 TRUE GTVK tested pool B*57:01 LW indi- re- vid- sultsually CU02 LVGP  41 not see CUO2_pool_1 Y chr8_143930249_G_A snp PLECP863L 528.5 HLA-  4  42   287.473059 TRUE AQLS tested pool B*57:01 HWindi- re- vid- sults ually CU02 QTAA  31 not see CU02_pool_1 Ychr7_77773271_A_G snp RSBN1L T584A  25.89 HLA-  5  19    76.1012011 TRUEAVGV tested pool A*03:01 LK indi- re- vid- sults ually CU02 FPSP  38 notsee Cu02_pool_1 Y chr12_132750694_G_T snp ANKLE2 P266T  43.78 HLA-  6 26   131.765585 TRUE SKTS tested pool B*07:02 LTL indi- re- vid- sultsually CU02 SSTS  42 not see CU02_pool_1 Y chr10_96604023_G_A snp PIK3AP1R733W   9.84 HLA-  7  30   162.029882 TRUE NRSS tested pool B*57:01 TWindi- re- vid- sults ually CU02 LVYG  32 not see CU02_pool_1 Ychr13_3821175_C_T snp RFC3 S44L   9.76 HLA-  8   2     8.21211585 TRUEPLGA tested pool A*03:01 GK indi- re- vid- sults ually CU02 HSYS  43 notsee CU02_pool_1 Y chr8_119802006_C_G snp TAF2 D194H  29.74 HLA-  9   3   10.120376 TRUE ELCT tested pool B*57:01 W indi- re- vid- sults uallyCU02 VTLD  44 not see CU02_pool_1 Y chr9_108979413_T_G snp CTNNAL1 E323D 32.44 HLA- 10 136  2107.24068 TRUE VILE tested pool B*57:01 R indi- re-vid- sults ually CU02 HSKP  45 not see CU02_pool_1 Y chr12_133057238_A_Gsnp ZNF84 T175A  29.84 HLA- 11  23    90.7546185 TRUE EDTD tested poolB*57:01 AW indi- re- vid- sults ually CU03 IAAS 179 not CU03_pool_1 Nchr1_230868472_G_A snp C1orf198 A14V  36.47 HLA-  0  19   146.699014TRUE RSVV tested C*12:03 M indi- vid- ually CU03 AAIA 180 not CU03_poo_1N chr1_230868472_G_A snp C1orf198 A14V  36.47 HLA-  2  42   492.404622TRUE ASRS tested C*12:03 V indi- vid- ually CU03 AASR 181 notCU03_pool_1 N chr1_230868472_G_A snp C1orf198 A14V  36.47 HLA-  6 116 3437.73836 TRUE SVVM tested C*12:03 indi- vid- ually CU03 EMDM 182 notCU03_pool_1 N chr5_37180032_T_A snp C5orf42 I1908L  14.78 HLA-  8   7   35.7275148 TRUE HLSD tested A*01:01 Y indi- vid- ually CU03 VENQ 183not CU03_pool_1 N chr12_30728769_C_T snp CAPRIN2 S554N   6.69 HLA- 10124  3970.47602 TRUE KHSL tested B*08:01 indi- vid- ually CU03 QYMD 184not CU03_pool_1 N chr10_60788061_G_T snp CDK1 S107I  26.84 HLA-  7   8   50.3301427 TRUE SSLV tested A*23:01 KI indi- vid- ually CU03 SASL 185not CU03_pool_1 N chr2_25929006_C_T snp KIF3C R785H  17.29 HLA-  9  30  260.370195 TRUE HPAT tested C*12:03 V indi- vid- ually CU03 VPDQ 186not CU03_pool_1 N chr6_63685063_T_G snp PHF3 N447K  47.53 HLA- 13 130 4071.14261 TRUE KSKQ tested B*08:01 L indi- vid- ually CU03 IVFI 187not CU03_pool_1 N chr11_65976483_A_T snp SART1 N554I  70.53 HLA-  5   3   17.4168253 TRUE ATSE tested B*15:03 F indi- vid- ually CU03 YPAP 188not CU03_pool_1 N chr20_44066022_C_A snp TOX2 S382Y  11.56 HLA- 11 101 2455.95947 TRUE QPPV tested B*08:01 L indi- vid- ually

Supplementary Table 4 Donor ID Analyte (average) Stimulus 1-038-001 CU041-024-001 1-024-002 CU02 Granzyme B (pg/ml)* DMSO 1786.73 1383.532639.03 854.78 1449.74 Peptide Pool 1 1672.60 4269.64 2449.23 1281.541132.49 DMSO 1874.02 3747.71 2382.01 626.20 n/a Peptide Pool 2 3118.303191.90 2006.73 872.89 n/a TNFalpha (pg/ml)^(#) DMSO 37.58 34.64 21.7638.07 1.22 Peptide Pool 1 53.02 217.57 42.05 57.13 7.44 DMSO 16.58 80.8124.98 24.77 n/a Peptide Pool 2 61.54 75.70 33.70 48.84 n/a IL-2(pg/ml)^(#) DMSO 1.78 3.86 4.24 0.23 6.67 Peptide Pool 1 15.53 9.88 7.750.00 0.00 DMSO 26.66 27.25 5.72 10.20 n/a Peptide Pool 2 0.00 19.1511.48 0.00 n/a IL-5 (pg/ml)^(#) DMSO 26.47 5.20 20.92 11.96 18.91Peptide Pool 1 10.48 14.65 26.72 9.42 17.64 DMSO 27.31 19.65 11.01 29.93n/a Peptide Pool 2 26.47 25.43 20.11 40.11 n/a Positive values are shownin italics. *Granzyme B ELISA: Values ≥1.5-fold over DMSO backgroundwere considered positive. ^(#)U-Plex MSD assay: Values ≥1.5-fold overDMSO background were considered positive

Supplementary Table 5TSNA and Infectious Disease Epitopes in IVS Control ExperimentsPredicted Origin Predicted Binding Mutation SEQ ID (Cell Line, HLAAffinity Mutation Nucleotide Peptide Name Sequence NO: Gene) Restriction(nM) Position Change Neoantigen_A1 APKKKSIKL 189 H2009 PPFIA3 B*07:02 125 chr19-49140014 C-to-T Neoantigen_A2 LLLEVVWHL 190 H128 FANCAA*02:01    6 chr16-89808348 C-to-T Neoantigen_A3 FTDEKVKAY 191H2122 PDE10A A*01:01   41 chr6-165543564 G-to-T Neoantigen_A6 RTAKQNPLTK192 H2122 GPR183 A*03:01  138 chr13-99295446 G-to-A Neoantigen_A7FLAPTGVPV 193 H128 NTM A*02:01    8 chr11-131911555 T-to-CNeoantigen_A10 RLADAEKLFQL 194 H128 PLEKHG4 A*02:01  201 chr16-67284435G-to-A Neoantigen A11 RTAKQNPLTKK 195 H2122 GP8183 A*03:01  131chr13-99295446 G-to-A Neoantigen_B2 IMYLTGMVNK 196 H2009 GSPT1 A*03:01  33 chr16-11891120 G-to-A Neoantigen_B3 TLQELSHAL 197 H128 PRPF19A*02:01  106 chr11-60902829 G-to-T Neoantigen_B6 VSQPVAPSY 198 Colo829A*01:01  948 chr1-35479047 C-to-T KIAA0319L Neoantigen_B7 RLFTPISAGY 199H2126 CYP26B1 A*03:01  157 chr2-72133060 G-to-C Neoantigen_B8 ITEEPILMTY200 H2122 RPL1L A*01:01  308 chr8-10611205 C-to-A Neoantigen_B10KVTGHRWLK 201 H2009 BSG A*03:01   51 chr19-579577 G-to-A Neoantigen_B12KLSEQILKK 202 H2009 TLR5 A*03:01   39 chr1-223110532 C-to-GNeoantigen_C3 GTKPNPHVY 203 H2126 OAS3 A*03:01 7336 chr12-112961105G-to-T Neoantigen_C4 QQQQVVTNK 204 H2126 LRP1 A*03:01 2361chr12-57162861 G-to-T Neoantigen_C5 KVLGKGSFAK 205 H2126 PLK2 A*03:01  40 chr5-58459089 G-to-A Neoantigen_C6 SVQAPVPPK 206 H2009 ENGASEA*03:01  279 chr17-79084548 C-to-G EBV RAKF RAKFKQLL 207 EBV BZLF-1B*08:01  457 Nan Nan Flu CTEL CTELKLSDY 208 Influenza NP A*01:01   39Nan Nan Flu ELRS ELRSRYWAI 209 Influenza A B*0801   12 Nan Nan CMV NLVPNLVPMVATV 210 CMV pp65 A*02:01   45 Nan Nan Flu GILG GILGFVFTL 211Influenza MP A*02:01   20 Nan Nan HCV KLVA KLVALGINAV 212 HCV NS3A*02:01   49 Nan Nan HIV ILKE ILKEPVHGV 213 HIV pol A*02:01  144 Nan NanRSV NPKA NPKASLLSL 214 RSV NP B*07:02   60 Nan Nan *Mutated NaN Nan Nanpeptides in neoantigen sequences are underlined. **Tumor cell NaN NanNan lines: Colo829, H128, H2009, H2122, H2126

Supplementary Table 6 TCR Nucleic Acid Sequences SEQ ID Consensus IDTCR Nucleic Acid Sequence NO: clonotype3_consensus_2GATTAGGCCAGTATGTGTAAGGGGCTGAACAGGCTTGCCATTGATTGGCTGGATAGGAAGGCCAGAAC 215TTCCTTCTAGGGGTAGAAGAACCCCAGTAACACCTATCAAACTAAACAGAATGGCTTTTTGGCTGAGAAGGCTGGGTCTACATTTCAGGCCACATTTGGGGAGACGAATGGAGTCATTCCTGGGAGGTGTTTTGCTGATTTTGTGGCTTCAAGTGGACTGGGTGAAGAGCCAAAAGATAGAACAGAATTCCGAGGCCCTGAACATTCAGGAGGGTAAAACGGCCACCCTGACCTGCAACTATACAAACTATTCTCCAGCATACTTACAGTGGTACCGACAAGATCCAGGAAGAGGCCCTGTTTTCTTGCTACTCATACGTGAAAATGAGAAAGAAAAAAGGAAAGAAAGACTGAAGGTCACCTTTGATACCACCCTTAAACAGAGTTTGTTTCATATCACAGCCTCCCAGCCTGCAGACTCAGCTACCTACCTCTGTGCTCTAAATGCCAGACTCATGTTTGGAGATGGAACTCAGCTGGTGGTGAAGCCCAATATCCAGAAGCCTGACCCTGCCGTGTACCAGCTGAGAGACTCCCCTGclonotype3_consensus_1GCCCATCTGTCGGTGAATTGAAAAGAAACAGAGCAAAATGACTCCTCCAATGGTGATGAGCCTGCCCC 216TGGGATTTGGAAACTTGGTAACAGAGAAAACCAATATAGACAAAGGATTTTAAACAGGATTATGGTCAATTAAGCAAATTAGAAAAGGATACTTGAAGGGGGATTTGGGACACAGGAGTCAAAAACACCGGGAAGACATGAGAAGTTTCCCTAAGAGTCTAAATTAGAGAAATATTTAGATAACTGACACCAGTGTATGAATGAGGAATTATATCATCACAGGGATAAGAGTTCCATTGAGTTACAAACTGCTTCCAAAAAGGTTAAGAAAAACTCGTAAGGCTGTGTCAATTCAGACAAAGGCATTCTTCCCATTCAAACGGTTCACCGGTGCATGAATCTTGAATTTGACCATCTGGGGAAGGGGCGTGGCCTCTCCTGACAGGAAGGCTCTGGGGCCCAGGCAGGGAGAATGAAGTCTCAGAATGACCCCCTTGAGAGTACTGTTCCCCTATCACCGATGCACAGACCCAGAAGACCCCTCCATCCTGTAGCACCTGCCATGAGCATCGGGCTCCTGTGCTGTGTGGCCTTTTCTCTCCTGTGGGCAAGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATAACTCCATGTACTGGTATCGACAAGACCCAGGCATGGGACTGAGGCTGATTTATTACTCAGCTTCTGAGGGTACCACTGACAAAGGAGAAGTCCCCAATGGCTACAATGTCTCCAGATTAAACAAACGGGAGTTCTCGCTCAGGCTGGAGTCGGCTGCTCCCTCCCAGACATCTGTGTACTTCTGTGCCAGCAGTTACCGGGGAGTACAACACTGAAGCTTTCTTTGGACAAGGCACCAGACTCACAGTTGTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGGAGTCTCTCAGCTGGTACACGGCAGGGTCAGGCTTCTGGATATTTGGTTGCACTTGGAGTCTTGTTCCACTCCCAAAAGTAAGTGCTCTCCTGCCCGTGACGGTCACAGCACAGAAGTACTCAGCCGCGTCGCTCATATGGGCTGAGGGTTTCGTCAGGTGGAAGGAGGTTTCACTCTTCTTAAATTCAGCCTCAAAACCGTTGATGCCTTTAACCAGGGTGGCCCCTGTTGTGTACTTCAGGAGAAGCTGGAGTCCTTGGTTGGGGTATTGCACATACCAGAAGAGATATGGTGGAACAGACGATGAGTAGTTGCACCTCAGCAGAACCAGGGCTCCCTCAGAGACAGAGACGTGGCTGCCAAGCTGGGTCACCGACTGGGCTCTGGTTCCTCCCAGGGTAAAAATCACCTCGAGCACTGGGACGAGCAGCAGGAGCATGGCTGAGCAGTGGCCACGCTGGAGGGCCCTGAGCAGAGCGGACAGAAGCCAAGGGCCCTGAGCCTCAGGAGCTAGGAGCAGCAGGCAGGTTAGGCTGGACAAGTCTCTGCCTTTGAAGGGTTTCAAAAATGGTTAAAGAAAGGCTTTCTCTTAAGGAGCCTGGATGTGTTCTGACCTCTTGTTCTCATCACAGCTCCCTATCTAC clonotype6_consensus_2GATCCTCATCCACTGAGCCTCCTCCCTGCAGCTGGCTGATGTAGCTCACTGGTGTCTGTGTAGATAGG 217GAGCTGTGATGAGAACAAGAGGTCAGAACACATCCAGGCTCCTTAAGAGAAAGCCTTTCTTTAACCATTTTTGAAACCCTTCAAAGGCAGAGACTTGTCCAGCCTAACCTGCCTGCTGCTCCTAGCTCCTGAGGCTCAGGGCCCTTGGCTTCTGTCCGCTCTGCTCAGGGCCCTCCAGCGTGGCCACTGCTCAGCCATGCTCCTGCTGCTCGTCCCAGTGCTCGAGGTGATTTTTACCCTGGGAGGAACCAGAGCCCAGTCGGTGACCCAGCTTGGCAGCCACGTCTCTGTCTCTGAGGGAGCCCTGGTTCTGCTGAGGTGCAACTACTCATCGTCTGTTCCACCATATCTCTTCTGGTATGTGCAATACCCCAACCAAGGACTCCAGCTTCTCCTGAAGTACACAACAGGGGCCACCCTGGTTAAAGGCATCAACGGTTTTGAGGCTGAATTTAAGAAGAGTGAAACCTCCTTCCACCTGACGAAACCCTCAGCCCATATGAGCGACGCGGCTGAGTACTTCTGTGCTGTGACCGTCACGGGCAGGAGAGCACTTACTTTTGGGAGTGGAACAAGACTCCAAGTGCAACCAAATATCCAGAAGCCTGACCCTGCCGTGTACCAGCTGAGAGACTAGATCGGAAC clonotype6_consensus3GGTTGCCAGAGACACCAGTAATTCTGCCAGACCTTGCCTGTGGGGCCATGGGAGCTCAAAATGCCCCT 218CCTTTCCTCCACAGGACCAGATGCCTGAGCTAGGAAAGGCCTCATTCCTGCTGTGATCCTGCCATGGATACCTGGCTCGTATGCTGGGCAATTTTTAGTCTCTTGAAAGCAGGACTCACAGAACCTGAAGTCACCCAGACTCCCAGCCATCAGGTCACACAGATGGGACAGGAAGTGATCTTGCGCTGTGTCCCCATCTCTAATCACTTATACTTCTATTGGTACAGACAAATCTTGGGGCAGAAAGTCGAGTTTTCTGGTTTCCTTTTATAATATGAAATCTCAGAGAAGTCTGAAATATTCGATGATCAATTCTCAGTTGAAAGGCCTGATGGATCAAATTTCACTCTGAAGATCCGGTCCACAAAGCTGGAGGACTCAGCCATGTACTTCTGTGCCAGCAACCCCCCGGACGCTGCGAGGGGACAAGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGGAGTCTCTCAGCTGGTACACGGCAGGGTCAGGCTTCTGGATATTTGGTTGCACTTGGAGTCTTGTTCCACTCCCAAAAGTAAGTGCTCTCCTGCCCGTGACGGTCACAGCACAGAAGTACTCAGCCGCGTCGCTCATATGGGCTGAGGGTTTCGTCAGGTGGAAGGAGGTTTCACTCTTCTTAAATTCAGCCTCAAAACCGTTGATGCCTTTAACCAGGGTGGCCCCTGTTGTGTACTTCAGGAGAAGCTGGAGTCCTTGGTTGGGGTATTGCACATACCAGAAGAGATATGGTGGAACAGACGATGAGTAGTTGCACCTCAGCAGAACCAGGGCTCCCTCAGAGACAGAGACGTGGCTGCCAAGCTGGGTCACCGACTGGGCTCTGGTTCCTCCCAGGGTAAAAATCACCTCGAGCACTGGGACGAGCAGCAGGAGCATGGCTGAGCAGTGGCCACGCTGGAGGGCCCTGAGCAGAGCGGACAGAAGCCAAGGGCCCTGAGCCTCAGGAGCTAGGAGCAGCAGGCAGGTTAGGCTGGACAAGTCTCTGCCTTTGAclonotype6_consensus_1GGGGCGTGGCCTCTCCTGACAGGAAGGCTCTGGGGCCCAGGCAGGGAGAATGAAGTCTCAGAATGACC 219CCCTTGAGAGTACTGTTCCCCTATCACCTGTAGCACCTGCCATGAGCATCGGGCTCCTGTGCTGTGTGGCCTTTTCTCTCCTGTGGGCAAGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATAACTCCATGTACTGGTATCGACAAGACCCAGGCATGGGACTGAGGCTGATTTATTACTCAGCTTCTGAGGGTACCACTGACAAAGGAGAAGTCCCCAATGGCTACAATGTCTCCAGATTAAACAAACGGGAGTTCTCGCTCAGGCTGGAGTCGGCTGCTCCCTCCCAGACATCTGTGTACTTCTGTGCCAGCAGTTACCGGGAGTACAACACTGAAGCTTTCTTTGGACAAGGCACCAGACTCACAGTTGTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGGAGTCTCTCAGCTGGTACACGGCAGGGTCAGGCTTCTGGATATTTGGTTGCACTTGGAGTCTTGTTCCACTCCCAAAAGTAAGTGCTCTCCTGCCCGTGACGGTCACAGCACAGAAGTACTCAGCCGCGTCGCTCATATGGGCTGAGGGTTTCGTCAGGTGGAAGGAGGTTTCACTCTCTTAAATTCAGCCTCAAAACCGTTGATGCCTTTAACCAGGGTGGCCCCTGTTGTGTACTTCAGGAGAAGCTGGAGTCCTTGGTTGGGGTATTGCACATACCAGAAGAGATATGGTGGAACAGACGATGAGTAGTTGCACCTCAGCAGAACCAGGGCTCCCTCAGAGACAGAGACGTGGCTGCCAAGCTGGGTCACCGACTGGGCTCTGGTTCCTCCCAGGGTAAAAATCACCTCGAGCACT clonotype10_consensus_1TGGGATTGAAAGGAAAGGACTGAGCTTGCCTGTGACTGGCTAGGGAGGAACCTGAGACTAGGGGACAG 220AAAGACTAGGGATTCACCCAGTAAAGAGAGCTCATCTGTGACTGAGGAGCCTTGCTCCATTTCAGGTCTTCTGTGATTTCAATAAGGAAGAAGAATGGAAACTCTCCTGGGAGTGTCTTTGGTGATTCTATGGCTTCAACTGGCTAGGGTGAACAGTCAACAGGGAGAAGAGGATCCTCAGGCCTTGAGCATCCAGGAGGGTGAAAATGCCACCATGAACTGCAGTTACAAAACTAGTATAAACAATTTACAGTGGTATAGACAAAATTCAGGTAGAGGCCTTGTCCACCTAATTTTAATACGTTCAAATGAAAGAGAGAAACACAGTGGAAGATTAAGAGTCACGCTTGACACTTCCAAGAAAAGCAGTTCCTTGTTGATCACGGCTTCCCGGGCAGCAGACACTGCTTCTTACTTCTGTGCTACGGCTAGCCGTCAGGGCGGATCTGAAAAGCTGGTCTTTGGAAAGGGAACGAAACTGACAGTAAACCCATATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTclonotype10_consensus_2GAGAGATACAGCATGAGACCTCCGGGTCCAGACAGCTCTGGAGCCCAAGGCGATGAGCCATGCATTGA 221TGTTGTTAAAAAGGAGCTGATAAATATTTAAAGCAGCACCCAACTGTGTTCTAATAGAAATGCTGTGATCCTGAGGTCCTGGGGATTGAGAGAGGAAGTGATGTCACTGTGGGAACTGCCCTGTGGAGACAAGGACATCCCTCATCCTCTGCTGCTGCTCACAGTGACACTGATCTGGTAAAGCCCTCATCCTGTCCTGACCCTGCCATGGGCACCAGTCTCCTATGCTGGGTGGTCCTGGGTTTCCTAGGGACAGATCACACAGGTGCTGGAGTCTCCCAGTCTCCCAGGTACAAAGTCACAAAGAGGGGACAGGATGTAGCTCTCAGGTGTGATCCAATTTCGGGTCATGTATCCCTTTATTGGTACCGACAGGCCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCAATTATGAAGCCCAACAAGACAAATCAGGGCTGCCCAATGATCGGTTCTCTGCAGAGAGGCCTGAGGGATCCATCTCCACTCTGACGATCCAGCGCACAGAGCAGCGGGACTCGGCCATGTATCGCTGTGCCAGCAGCCGAGGGGGGGGCACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAACCAAATATCCAGAAGCCTGACCCTGCCGTGTACCAGCTGAGAGACT clonotype1_consensus_1GGGACATTTCTCAAATGAGAAGCAAACAGTTCACTTCCTTGGATCCGTGGTTTCGCCTGTGGCCTTCA 222GGGGGCGACGTTGCACTAAGGAGGCATCTGTGTTCATTGCCGACCATCCTCATCCACTGAGCCTCCTCCCTGCAGCTGGCTGATGTAGCTCACTGGTGTCTGTGTAGATAGGGAGCTGTGATGAGAACAAGAGGTCAGAACACATCCAGGCTCCTTAAGAGAAAGCCTTTCTTTAACCATTTTTGAAACCCTTCAAAGGCAGAGACTTGTCCAGCCTAACCTGCCTGCTGCTCCTAGCTCCTGAGGCTCAGGGCCCTTGGCTTCTGTCCGCTCTGCTCAGGGCCCTCCAGCGTGGCCACTGCTCAGCCATGCTCCTGCTGCTCGTCCCAGTGCTCGAGGTGATTTTTACCCTGGGAGGAACCAGAGCCCAGTCGGTGACCCAGCTTGGCAGCCACGTCTCTGTCTCTGAGGGAGCCCTGGTTCTGCTGAGGTGCAACTACTCATCGTCTGTTCCACCATATCTCTTCTGGTATGTGCAATACCCCAACCAAGGACTCCAGCTTCTCCTGAAGTACACAACAGGGGCCACCCTGGTTAAAGGCATCAACGGTTTTGAGGCTGAATTTAAGAAGAGTGAAACCTCCTTCCACCTGACGAAACCCTCAGCCCATATGAGCGACGCGGCTGAGTACTTCTGTGCTGTGACCGTCACGGGCAGGAGAGCACTTACTTTTGGGAGTGGAACAAGACTCCAAGTGCAACCAAATATCCAGAAGCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTGATGGCTCAAACACAGCGACCTCGGGTGGGAACACGTTTTTCAGGTCCTCGAGCACCAGGAGCCGCGTGCCTGGCCCGAAGTACTGGGTCTCTTGTCCCCTCGCAGCGTCCGGGGGGTTGCTGGCACAGAAGTACATGGCTGAGTCCTCCAGCTTTGTGGACCGGATCTTCAGAGTGAAATTTGATCCATCAGGCCTTTCAACTGAGAATTGATCATCGAATATTTCAGACTTCTCTGAGATTTCATTATTATAAAAGGAAACCAGAAACTCGACTTTCTGCCCCAAGATTTGTCTGTACCAATAGAAGTATAAGTGATTAGAGATGGGGACACAGCGCAAGATCACTTCCTGTCCCATCTGTGTGACCTGATGGCTGGGAGTCTGGGTGACTTCAGGTTCTGTGAGTCCTGCTTTCAAGAGACTAAAAATTGCCCAGCATACGAG clonotype1_consensus_2AAACGATTTCTTATATGGGGAGAGGGCGCTCCAGGGATGGTGGGTGTTGCCAGAGACACCAGTATTCT 223GACCAGATGCCTGAGCTAGGAAAGGCCTCATTCCTGCTGTGATCCTGCCATGGATACCTGGCTCGTATGCTGGGCAATTTTTAGTCTCTTGAAAGCCCCAGCCATCAGGTCACACAGATGGGACAGGAAGTGATCTTGCGCTGTGTCCCCATCTCTAATCACTTATACTTCTATTGGTACAGACAAATCTTGAATAATGAAATCTCAGAGAAGTCTGAAATATTCGATGATCAATTCTCAGTTGAAAGGCCTGATGGATCAAATTTCACTCTGAAGATCCGGTCCACACCAGCAACCCCCCGGACGCTGCGAGGGGACAAGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAGGACCTGAAAAACGTGTTCCGAGTCTCTCAGCTGGTACACGGCAGGGTCAGGCTTCTGGATATTTGGTTGCACTTGGAGTCTTGTTCCACTCCCAAAAGTAAGTGCTCTCCTGCCCGTGACGGTCACAGCACAGAAGTACTCAGCCGCGTCGCTCATATGGGCTGAGGGTTTCGTCAGGTGGAAGGAGGTTTCACTCTTCTTAAATTCAGCCTCAAAACCGTTGATGCCTTTAACCAGGGTGGCCCCTGTTGTGTACTTCAGGAGAAGCTGGAGTCCTTGGTTGGGGTATTGCACATACCAGAAGAGATATGGTGGAACAGACGATGAGTAGTTGCACCTCAGCAGAACCAGGGCTCCCTCAGAGACAGAGACGTGGCTGCCAAGCTGGGTCACCGACTGGGCTCTGGTTCCTCCCAGGGTAAAAATCACCTCGAGCACTGGGACGAGCAGCAGGAGCATGGCTGAGCAGTGGCCACGCTGGAGGGCCCTGAGCAGAGCGTCAGGAGCTAGGAGCAGCAGGCAGGTTAGGCTGGACAAGTCTCTGCCTTTGAAGGGTTTCAAAAATGGTTAAAGAAAGGCTTTCTCTTAAGGAGCCTGGATGTGTTCTGACCTCTTGTTCTCATCACAGCTCCCTATCTACACAGACACCAGTGAGCTACATCAGCCAGCTGCAGGGAGGAGGCTCAGTGGAT clonotype4_consensus_1GCTGATGGCAGCAGGATGATGTTACGATCACAAGAGGGCAGTGCCTCCCTCTCCTCAGACTAACTTGG 224CTTTAAATCAGCCTATCTGCATTGAAAGGGAGGAACCTGAGACTAGGGGACAGAAAGACTAGGGATTCACCCAGTAAAGAGAGCTCATCTGTGACTGAGGAGCCTTGCTCCATTTCAGGTCTTCACTCTCCTGGGAGTGTCTTTGGTGATTCTATGGCTTCAACTGGCTAGGGTGAACAGTCAACAGGGAGAAGAGGATCCTCAGGCCTTGAGCATCCAGTACAAAACTAGTATAAACAATTTACAGTGGTATAGACAAAATTCAGGTAGAGGCCTTGTCCACCTAATTTTAATACGTTCAAATGAAAGAGAGAAACACAGTGGAAGATTAAGAGTCACGCTTGACACTTCCAAGAAAAGCAGTTCCTTGTTGATCACGGCTTCCCGGGCAGCAGACACTGCTTCTTACTTCTGTGCTACGGGCCTAGATTTGGACAAGCTCATCTTTGGGACTGGGACCAGATTACAAGTCTTTCCAAATATCCAGAAGCCTGACCCTGCCGTGTACCAGCTGAGAGACTAGAT clonotype4_consensus_2GGGGCAATGTGGGTGTTATACTGAAAAGATCACAGATGGTTCACTTTGCAAGTAAAACTGTAAATGTT 225CTTAAGTGTGCATTTCTGCTGCTTCTGATGGGCTGAAAATCCCCTTTGATTTCTAAAGTAAATGTAGAGACGTTTTAAAAATAAAGGACTCCTTTGTCCAAGATATATTCCGAAATCCTCCAACAGAGACCTGTGTGAGCTTCTGCTGCAGTAATGGTGAAGATCCGGCAATTTTGTTGGCTATTTTGTGGCTTCAGCTAAGCTGTGTAAGTGCCGCCAAAAATGAAGTGGAGCAGAGTCCTCAGAACCTGACTGCCCAGGAAGGAGAATTTATCACAATCAACTGCAGTTACTCGGTAGGAATAAGTGCCTTACACTGGCTGCAACAGCATCCAGGAGGAGGCATTGTTTCCTTGTTTATGCTGAGCTCAGGGAAGAAGAAGCATGGAAGATTAATTGCCACAATAAACATACAGGAAAAGCACAGCTCCCTGCACATCACAGCCTCCCATCCCAGAGACTCTGCCGTCTACATCTGTGCTGTCAGATGGGGCGGTAACCAGTTCTATTTTGGGACAGGGACAAGTTTGACGGTCATTCCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTAGATCGGAAGCAclonotype4_consensus_3GTGGAAACCCACTTCTGACTTATCACTTGTCATGAATTCTATGCTTCATGGTGTTACACCGTTTATTG 226TTTCTGATGAGTGACAGTAATTATTTTCTTTCTTGCTGGTACATAATAAAGTGGTGCACATCAGAGTTGCTGCCATCTTAGACTTAACTCATCAGTATCAGGTGATCCTGAGGCTCAGTGATGTCACTGTGGGAACTGCTCTGTGGCGACAAGGACGTCCCTCATCCTCTGCTCCTGCTCACAGTGACCCTGATCTGGTAAAGCTCCCATCCTGCCCTGACCCTGCCATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGGGGGCAGATCACGCAGATACTGGAGTCTCCCAGGACCCCAGACACAAGATCACAAAGAGGGGACAGAATGTAACTTTCAGGTGTGATCCAATTTCTGAACACAACCGCCTTTATTTGGTACCGACAGACCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGAAGCTCAACTAGAAAAATCAAGGCTGCTCAGTGATCGGTTCTCTGCAGAGAGGCCTAAGGGATCTTTCTCCACCTTGGAGATCCAGCGCACAGAGCAGGGGGACTCGGCCATGTATCTCTGTGCCAGCAGCTTAGCCGGGACAGGGGGTAATTATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGGAGTCTCTCAGCTGGTACACGGCAGGGTCAGGCTTCTGGATATTTGGTTGCACTTGGAGTCTTGTTCCACTCCCAAAAGTAAGTGCTCTCCTGCCCGTGACGGTCACAGCAGGCTGAGGGTTTCGTCAGGTGGAAGGAGGTTTCACTCTTCTTAAATTCAGCCTCAAAACCGTTGATGCCTTTAACCAGGGTGGCCCCTGTTGTGTATATTGCACATACCAGAAGAGATATGGTGGAACAGACGATGAGTAGTTGCACCTCAGCAGAACCAGGGCTCCCTCAGAGACAGAGACGTGGCTGCCAAGCTGGGTCACCGACTGGGCTCTGGTTCCTCCCAGGGTAAAAATCACCTCGAGCACTGGGACGAGCAGCAGGAGCATGGCTGAGCAGTGGCCACGCTGGAGGGCCCTGAGCAGAGCGGACAGAAGCCAAGGGCCCTGAGCCTCAGGAGCTAGGAGCAGCAGGCAGGTTAGGCTGGACAAGTCTCTGCCTTTGAAGGGTTT clonotype9_consensus_1GGGAGGCTTTGTCGGGTGGAGCTGATTGGTTGCAGGAGCAGCAACAGTTCCAGAGCCAAGTCATGACA 227CCGACCTCCCCAAGGTTTAGTTAAATATATCTTATGGTGAAAATGCCCGGAGCAAGAAGGCAAAGCATCATGAAGAGGATATTGGGAGCTCTGCTGGGGCTCTTGAGTGCCCAGGTTTGCTGTGTGAGAGGAATACAAGTGGAGCAGAGTCCTCCAGACCTGATTCTCCAGGAGGGAGCCAATTCCACGCTGCGGTGCAATTTTTCTGACTCTGTGAACAATTTGCAGTGGTTTCATCAAAACCCTTGGGGACAGCTCATCAACCTGTTTTACATTCCCTCAGGGACAAAACAGAATGGAAGATTAAGCGCCACGACTGTCGCTACGGAACGCTACAGCTTATTGTACATTTCCTCTTCCCAGACCACAGACTCAGGCGTTTATTTCTGTGCTGTGGTGTTGGATAGCAACTATCAGTTAATCTGGGGCGCTGGGACCAAGCTAATTATAAAGCCAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTC clonotype9_consensus_2GGGAGTCATCCCTCCTCGCTGGTGAATGGAGGCAGTGGTCACAACTCTCCCCAGAGAAGGTGGTGTGA 228GGCCATCACGGAAGATGCTGCTGCTTCTGCTGCTTCTGGGGCCAGGCTCCGGGCTTGGTGCTGTCGTCTCTCAACATCCGAGCAGGGTTATCTGTAAGAGTGGAACCTCTGTGAAGATCGAGTGCCGTTCCCTGGACTTTAGGCCACAACTATGTTTTGGTATCGTCAGTTCCCGAAACAGAGTCTCATGCTGATGGCAACTTCCAATGAGGGCTCCAAGGCCACATACGAGCAAGGCGTCGAGAAGGACAAGTTTCTCATCAACCATGCAAGCCTGACCTTGTCCACTCTGACAGTGACCAGTGCCCATCCTGAAGACAGCAGCTTCTACATCTGCAGTGCAACCAGGGGGCACTTGAGCAAATCAGCCCCAGCATTTTGGTGATGGGACTCGACTCTCCATCCTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGGAGTCTCTCAGCTGGTACACGGCAGGGTCAGGCTTCTGGATATTTGGTTGCACTTGGAGTCTTGTTCCACTCCCAAAAGTAAGTGCTCTCCTGCCCGTGACGGTCACAGCACAGAAGTACTCAGCCGCGTCGCTCATATGGGCTGAGGGTTTCGTCAGGTGGAAGGAGGTTTCACTCTTCTTAAATTCAGCCTCAAAACCGTTGATGCCTTTAACCAGGGTGGCCCCTGTTGTGTACTTCAGGAGAAGCTGGAGTCCTTGGTTGGGGTATTGCACATACCAGAAGAGATATGGTGGAACAGACGATGAGTAGTTGCACCTCAGCAGAACCAGGGCTCCCTCAGAGACAGAGACGTGGCTGCCAAGCTGGGTCACCGACTGGGCTCTGGTTCCTCCCAGGGTAAAAATCACCTCGAGCACTGGGACGAGCAGCAGGAGCATGGCTGAGCAGTGGCCACGCTGGAGGGCCCTGAGCAGAGCGGACAGAAGCCAAGGGCCCTGAGCCTCAGGAGCTAGGAGCAGCAGGCAGGTTAGGCTGGACAAGTCTCTGCCTTTGAAGGGTTTCAAAAATGGTTA

Supplementary Table 7 Frequencies and CDR3a/b Sequences Clono- Fre- SEQSEQ type quen- ID ID ID cy Proportion cdr3s_aa NOS cdr3s_nt NOS clono-386 0.49171915 TRA:CAVTVTGRRALTF;  47 &TRA:TGTGCTGTGACCGTCACGGGCAGGAGAGCACTTACTTTT; 238 & type1TRB:CASNPPDAARGQET  48 TRB:TGTGCCAGCAACCCCCCGGACGCTGCGAGGGGACAAGA 239QYF GACCCAGTACTTC clono-  53 0.06751592 TRA:CALNARLMF; 229 &TRA:TGTGCTCTAAATGCCAGACTCATGTTT; 240 & type3 TRB:CASSYREYNTEAFF 230TRB:TGTGCCAGCAGTTACCGSSAGTACAACACTGAAGCTTTCT 241 TT clono-  34 0.0433121TRA:CATGLDLDKLIF; 231 & TRA:TGTGCTACGGGCCTAGATTTGGACAAGCTCATCTTT; 242 &type4 TRA:CAVRWGGNQFVF; 232 & TRA:TGTGCTGTCAGATGGGGCGGTAACCAGTTCTATTTT;243 & TRB:CASSLAGTGGNYE 233 TRB:TGTGCCAGCAGCTTAGCCGGGACAGGGGGTAATTATG244 QFF AGCAGTTCTTC clono-  10 0.01273885 TRA:CAVTVTGRRALTF;  47 &TRA:TGTGCTGTGACCGTCACGGGCAGGAGAGCACTTACTTTT; 238 & type6TRB:CASNPPDAARGQET  48 & TRB:TGTGCCAGCAACCCCCCGGACGCTGCGAGGGGACAAGAGA239 & QYF; CCCAGTACTTC; TRB:CASSYREYNTEAFF 230TRB:TGTGCCAGCAGTTACCGGGAGTACAACACTGAAGCTTTCT 241 TT clono-   7 0.0089172TRA:CAVVLDSNYQLIW; 234 & TRA:TGTGCTGTGGTGTTGGATAGCAACTATCAGTTAATCTGG;245 & type9 TRB:CSATRGHLSNQPQH 235TRB:TGCAGTGCAACCAGGGGGCACTTGAGCAATCAGCCCCAGC 246 F ATTTT clono-   50.00636943 TRA:CATASRQGGSEKLV 236 &TRA:TGTGCTACGGCTAGCCGTCAGGGCGGATCTGAAAAGCTGG 247 & type10 F; TCTTT;TRB:CASSRGGGTDTQYF 237 TRB:TGTGCCAGCAGCCGAGGGGGGGGCACAGATACGCAGTATT 248TT

Supplementary Table 8 Sequences and V, D, J Genes SEQ SEQ Clono- C FULLPro- ID ID type ID Consensus ID Length Chain V Gene D Gene J Gene GeneLength ductive cdr3 NO: cdr3_nt NO: Reads Umis   0 clono-clonotype1_consensus_1 1263 TRA TRAV8-4 None TRAJ5 TRAC TRUE TRUECAVTVTGRRALTF  47 TGTGCTGTGACCGTCACGGGCAG 238 2717775 10562 type1GAGAGCACTTACTTTT   1 clono- clonotype1_consensus_2 1332 TRB TRVB2 TRBD2TRBJ2-5 TRBC2 TRUE TRUE CASNPPDAARGQE  48 TGTGCCAGCAACCCCCCGGACGC 2391796338  7242 type1 TQYF TGCGAGGGGACAAGAGACCCAGT ACTTC   2 clono-clonotype10_consensus_1  604 TRA TRAV17 None TRAJ57 TRAC TRUE TRUECATASRQGGSEKL 236 TGTGCTACGGCTAGCCGTCAGGG 247    4006    13 type10 VFCGGATCTGAAAAGCTGGTCTTT   3 clono- clonotype10_consensus_2  774 TRBTRBV7-6 TRBD1 TRBJ2-3 TRBC2 TRUE TRUE CASSRGGGTDTQY 237TGTGCCAGCAGCCGAGGGGGGGGT 248    9401    35 type10 F CACAGAACGCAGTATTTT103 clono- clonotype3_consensus_1 1663 TRB TRBV6-1 TREBD2 TRBJ1-1 TRBC1TRUE TRUE CASSYREYNTEAF 230 TGTGCCAGCAGTTACCGGGAGTAT 241  243324   894type3 F CAACACGAAGCTTTCTTT 104 clono- clonotype3_conSensus_2  608 TRATRAV6 None TRAJ31 TRAC TRUE TRUE CALNARLMF 229 TGTGCTCTAAATGCCAGACTCATG240   87200   247 type3 TTT 125 clono- clonotype4_consensus_1  683 TRATRAV17 None TRAJ34 TRAC TRUE TRUE CATGLDLDKLIF 231TGTGCTACGGGCCTAGATTTGGAC 242  131803   385 type4 AAGCTCATCTTT 126 clono-clonotype4_consensus_2  669 TRA TRAV41 None TRAJ49 TRAC TRUE TRUECAVRWGGNQFYF 232 TGTGCTGTCAGATGGGGCGGTAAC 243   88320   307 type4CAGTTCTATTTT 127 clono- clonotype4_consensus_3 1315 TRB TRBV7-9 TRBD1TRBJ2-1 TRBC2 TRUE TRUE CASSLAGTGGNYE 233 TGTGCCAGCAGCTTAGCCGGGACA 244 237200   830 type4 QFF GGGGGTAATTATGAGCAGTTCTTC 171 clono-clonotype6_consensus_1 1007 TRB TRBV6-1 TRBD2 TRBJ1-1 TRBC1 TRUE TRUECASSYREYNTEAF 230 TGTGCCAGCAGTTACCGGGAGTAC 241   23803   105 type6 FAACACTGAAGCTTTCTTT 172 clono- clonotype6_consensus_2  713 TRA TRAV8-4None TRAJ5 TRAC TRUE TRUE CAVTVTGRRALTF  47 TGTGCTGTGACCGTCACGGGCAGG 238  24293   106 type6 AGAGCACTTACTTTT 173 clono- clonotype6_consensus_31147 TRB TRBV2 TRbD2 TRBj2-5 TRBC2 TRUE TRUE CASNPPDAARGQE  48TGTGCCAGCAACCCCCCGGACGCT 239   34437   132 type6 TQYFGCGAGGGGACAAGAGACCCAGTAC TFC 242 clono- clonotype9_consensus_1  568 TRATRAV22 None TRAJ33 TRAC TRUE TRUE CAVVLDSNYQLIW 234TGTGCTGTGGTGTTGGATAGCAAC 245    9883    29 type9 TATCAGTTAATCTGG 243clono- clonotype9_consensus_2 1102 TRB TRBV20-1 TRBD1 TRBJ1-5 TRBC1 TRUETRUE CSATRGHLSNQPQ 235 TGCAGTGCAACCAGGGGGCACTTG 246   38619   126 type9HF AGCAATCAGCCCCAGCATTTT

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1. A method for identifying one or more T-cells that areantigen-specific for at least one neoantigen from one or more tumorcells of a subject that are likely to be presented on surface of thetumor cells, the method comprising the steps of: obtaining at least oneof exome, transcriptome, or whole genome nucleotide sequencing data fromthe tumor cells and normal cells of the subject, wherein the nucleotidesequencing data is used to obtain data representing peptide sequences ofeach of a set of neoantigens identified by comparing the nucleotidesequencing data from the tumor cells and the nucleotide sequencing datafrom the normal cells, wherein the peptide sequence of each neoantigencomprises at least one alteration that makes it distinct from thecorresponding wild-type peptide sequence identified from the normalcells of the subject; encoding the peptide sequences of each of theneoantigens into a corresponding numerical vector, each numerical vectorincluding information regarding a plurality of amino acids that make upthe peptide sequence and a set of positions of the amino acids in thepeptide sequence; inputting the numerical vectors, using a computerprocessor, into a machine-learned presentation model to generate a setof presentation likelihoods for the set of neoantigens, eachpresentation likelihood in the set representing the likelihood that acorresponding neoantigen is presented by one or more MHC alleles on thesurface of the tumor cells of the subject, the machine-learnedpresentation model comprising: a plurality of parameters identified atleast based on a training data set comprising: for each sample in aplurality of samples, a label obtained by mass spectrometry measuringpresence of peptides bound to at least one MHC allele in a set of MHCalleles identified as present in the sample; and for each of thesamples, training peptide sequences encoded as numerical vectorsincluding information regarding a plurality of amino acids that make upthe peptides and a set of positions of the amino acids in the peptides;a function representing a relation between the numerical vector receivedas input and the presentation likelihood generated as output based onthe numerical vector and the parameters; selecting a subset of the setof neoantigens based on the set of presentation likelihoods to generatea set of selected neoantigens; identifying one or more T-cells that areantigen-specific for at least one of the neoantigens in the subset; andreturning the one or more identified T-cells.
 2. The method of claim 1,wherein inputting the numerical vector into the machine-learnedpresentation model comprises: applying the machine-learned presentationmodel to the peptide sequence of the neoantigen to generate a dependencyscore for each of the one or more MHC alleles indicating whether the MHCallele will present the neoantigen based on the particular amino acidsat the particular positions of the peptide sequence.
 3. The method ofclaim 2, wherein inputting the numerical vector into the machine-learnedpresentation model further comprises: transforming the dependency scoresto generate a corresponding per-allele likelihood for each MHC alleleindicating a likelihood that the corresponding MHC allele will presentthe corresponding neoantigen; and combining the per-allele likelihoodsto generate the presentation likelihood of the neoantigen.
 4. The methodof claim 3, wherein the transforming the dependency scores models thepresentation of the neoantigen as mutually exclusive across the one ormore MHC alleles.
 5. The method of claim 2, wherein inputting thenumerical vector into the machine-learned presentation model furthercomprises: transforming a combination of the dependency scores togenerate the presentation likelihood, wherein transforming thecombination of the dependency scores models the presentation of theneoantigen as interfering between the one or more MHC alleles.
 6. Themethod of claim 2, wherein the set of presentation likelihoods arefurther identified by at least one or more allele noninteractingfeatures, and further comprising: applying the machine-learnedpresentation model to the allele noninteracting features to generate adependency score for the allele noninteracting features indicatingwhether the peptide sequence of the corresponding neoantigen will bepresented based on the allele noninteracting features.
 7. The method ofclaim 6, further comprising: combining the dependency score for each MHCallele in the one or more MHC alleles with the dependency score for theallele noninteracting features; transforming the combined dependencyscores for each MHC allele to generate a per-allele likelihood for eachMHC allele indicating a likelihood that the corresponding MHC allelewill present the corresponding neoantigen; and combining the per-allelelikelihoods to generate the presentation likelihood.
 8. The method ofclaim 6, further comprising: combining the dependency scores for each ofthe MHC alleles and the dependency score for the allele noninteractingfeatures; and transforming the combined dependency scores to generatethe presentation likelihood.
 9. The method of claim 1, wherein the oneor more MHC alleles include two or more different MHC alleles.
 10. Themethod of claim 1, wherein the peptide sequences comprise peptidesequences having lengths other than 9 amino acids.
 11. The method ofclaim 1, wherein encoding the peptide sequence comprises encoding thepeptide sequence using a one-hot encoding scheme.
 12. The method ofclaim 1, wherein the plurality of samples comprise at least one of: (a)one or more cell lines engineered to express a single MHC allele; (b)one or more cell lines engineered to express a plurality of MHC alleles;(c) one or more human cell lines obtained or derived from a plurality ofpatients; (d) fresh or frozen tumor samples obtained from a plurality ofpatients; and (e) fresh or frozen tissue samples obtained from aplurality of patients.
 13. The method of claim 1, wherein the trainingdata set further comprises at least one of: (a) data associated withpeptide-MHC binding affinity measurements for at least one of thepeptides; and (b) data associated with peptide-MHC binding stabilitymeasurements for at least one of the peptides.
 14. The method of claim1, wherein the set of presentation likelihoods are further identified byat least expression levels of the one or more MHC alleles in thesubject, as measured by RNA-seq or mass spectrometry.
 15. The method ofclaim 1, wherein the set of presentation likelihoods are furtheridentified by features comprising at least one of: (a) predictedaffinity between a neoantigen in the set of neoantigens and the one ormore MHC alleles; and (b) predicted stability of the neoantigen encodedpeptide-MHC complex.
 16. The method of claim 1, wherein the set ofnumerical likelihoods are further identified by features comprising atleast one of: (a) the C-terminal sequences flanking the neoantigenencoded peptide sequence within its source protein sequence; and (b) theN-terminal sequences flanking the neoantigen encoded peptide sequencewithin its source protein sequence.
 17. The method of claim 1, whereinselecting the set of selected neoantigens comprises selectingneoantigens that have an increased likelihood of being presented on thetumor cell surface relative to unselected neoantigens based on themachine-learned presentation model.
 18. The method of claim 1, whereinselecting the set of selected neoantigens comprises selectingneoantigens that have an increased likelihood of being capable ofinducing a tumor-specific immune response in the subject relative tounselected neoantigens based on the machine-learned presentation model.19. The method of claim 1, wherein selecting the set of selectedneoantigens comprises selecting neoantigens that have an increasedlikelihood of being capable of being presented to naive T-cells byprofessional antigen presenting cells (APCs) relative to unselectedneoantigens based on the presentation model, optionally wherein the APCis a dendritic cell (DC).
 20. The method of claim 1, wherein selectingthe set of selected neoantigens comprises selecting neoantigens thathave a decreased likelihood of being subject to inhibition via centralor peripheral tolerance relative to unselected neoantigens based on themachine-learned presentation model.
 21. The method of claim 1, whereinselecting the set of selected neoantigens comprises selectingneoantigens that have a decreased likelihood of being capable ofinducing an autoimmune response to normal tissue in the subject relativeto unselected neoantigens based on the machine-learned presentationmodel.
 22. The method of claim 1, wherein the one or more tumor cellsare selected from the group consisting of: lung cancer, melanoma, breastcancer, ovarian cancer, prostate cancer, kidney cancer, gastric cancer,colon cancer, testicular cancer, head and neck cancer, pancreaticcancer, brain cancer, B-cell lymphoma, acute myelogenous leukemia,chronic myelogenous leukemia, chronic lymphocytic leukemia, and T-celllymphocytic leukemia, non-small cell lung cancer, and small cell lungcancer.
 23. The method of claim 1, further comprising generating anoutput for constructing a personalized cancer vaccine from the set ofselected neoantigens.
 24. The method of claim 23, wherein the output forthe personalized cancer vaccine comprises at least one peptide sequenceor at least one nucleotide sequence encoding the set of selectedneoantigens.
 25. The method of claim 1, wherein the machine-learnedpresentation model is a neural network model.
 26. The method of claim25, wherein the neural network model includes a plurality of networkmodels for the MHC alleles, each network model assigned to acorresponding MHC allele of the MHC alleles and including a series ofnodes arranged in one or more layers.
 27. The method of claim 26,wherein the neural network model is trained by updating the parametersof the neural network model, and wherein the parameters of at least twonetwork models are jointly updated for at least one training iteration.28. The method of claim 25, wherein the machine-learned presentationmodel is a deep learning model that includes one or more layers ofnodes.
 29. The method of claim 1, wherein identifying the one or moreT-cells comprises co-culturing the one or more T-cells with one or moreof the neoantigens in the subset under conditions that expand the one ormore T-cells.
 30. The method of claim 1, wherein identifying the one ormore T-cells comprises contacting the one or more T-cells with an MHCmultimer comprising one or more of the neoantigens in the subset underconditions that allow binding between the T-cells and the MHC multimer.31. The method of claim 1, further comprising identifying one or moreT-cell receptors (TCR) of the one or more identified T-cells.
 32. Themethod of claim 31, wherein identifying the one or more T-cell receptorscomprises sequencing the T-cell receptor sequences of the one or moreidentified T-cells.
 33. An isolated T-cell that is antigen-specific forat least one selected neoantigen in the subset of any-ene-e-claim
 1. 34.The method of claim 32, further comprising: genetically engineering aplurality of T-cells to express at least one of the one or moreidentified T-cell receptors; culturing the plurality of T-cells underconditions that expand the plurality of T-cells; and infusing theexpanded T-cells into the subject.
 35. The method of claim 34, whereingenetically engineering the plurality of T-cells to express at least oneof the one or more identified T-cell receptors comprises: cloning theT-cell receptor sequences of the one or more identified T-cells into anexpression vector; and transfecting each of the plurality of T-cellswith the expression vector.
 36. The method of claim 1, furthercomprising: culturing the one or more identified T-cells underconditions that expand the one or more identified T-cells; and infusingthe expanded T-cells into the subject.
 37. The method of claim 1,wherein the one or more T-cells that are antigen-specific for at leastone of the neoantigens in the subset are identified using between 5-30mL of whole blood from the subject.
 38. The method of claim 1, whereinthe subset of neoantigens comprises at most 20 neoantigens and whereinthe one of more identified T-cells recognize at least 2 neoantigens inthe subset of neoantigens.
 39. The method of claim 1, wherein the one ormore MHC alleles are class I MHC alleles.